Treatment of cancer by manipulation of commensal microflora

ABSTRACT

Provided herein are methods of treatment and/or prevention of cancer by manipulation of commensal microflora. In particular, the amount, identity, presence, and/or ratio of microflora (e.g., gut microflora) in a subject is manipulated to facilitate one or more co-treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 17/192,519, filed Mar. 4, 2021, which is a continuation of U.S.patent application Ser. No. 16/942,496, filed Jul. 29, 2020, nowabandoned, which is a continuation of U.S. patent application Ser. No.16/795,226, filed Feb. 19, 2020, now abandoned, which is a continuationof U.S. patent application Ser. No. 16/437,972, filed Jun. 11, 2019, nowabandoned, which is a continuation of U.S. patent application Ser. No.16/282,517, filed Feb. 22, 2019, now abandoned, which is a continuationof U.S. patent application Ser. No. 15/718,735, filed Sep. 28, 2017, nowabandoned, which is a continuation of U.S. patent application Ser. No.15/170,284, filed Jun. 1, 2016, now U.S. Pat. No. 9,855,302, whichclaims the priority benefit of U.S. Provisional Patent Application62/169,112, filed Jun. 1, 2015, and U.S. Provisional Patent Application62/248,741, filed Oct. 30, 2015, each of which is incorporated byreference in its entirety.

FIELD

Provided herein are methods of treatment and/or prevention of cancer bymanipulation of commensal microflora. In particular, the amount,identity, presence, and/or ratio of microflora (e.g., gut microflora) ina subject is manipulated to facilitate one or more co-treatments.

BACKGROUND

Harnessing the host immune system constitutes a promising approach forthe treatment of cancer because of its potential to specifically targettumor cells while limiting harm to normal tissue, with durability ofbenefit associated with immunologic memory. Enthusiasm has been fueledby recent clinical success, particularly with antibodies that blockimmune inhibitory pathways, specifically CTLA-4 and the PD-1/PD-L1 axis(Hodi et al. The New England journal of medicine 363, 711-723 (2010).;Hamid et al. The New England journal of medicine 369, 134-144 (2013).;herein incorporated by reference in their entireties). Early data haveindicated that clinical responses to these immunotherapies are morefrequent in patients who show evidence of an endogenous T cell responseongoing in the tumor microenvironment at baseline (Tumeh et al. Nature515, 568-571 (2014).; Spranger et al. Science translational medicine 5,200ra116 (2013).; Ji et al. Cancer immunology, immunotherapy: CII 61,1019-1031 (2012).; Gajewski et al. Cancer journal 16, 399-403 (2010).;herein incorporated by reference in their entireties). Despite thefunctional and clinical importance of this T cell-inflamed tumormicroenvironment, the mechanisms that govern the presence or absence ofthis phenotype have not been well understood. Theoretical sources ofinter-patient heterogeneity include germline genetic differences at thelevel of the host, variability in patterns of somatic alterations intumor cells, and environmental differences with the potential to impacton systemic immunity.

SUMMARY

Provided herein are methods of treatment and/or prevention of cancer bymanipulation of commensal microflora. In particular, the amount,identity, presence, and/or ratio of microflora (e.g., gut microflora) ina subject is manipulated to facilitate one or more co-treatments.

In some embodiments, provided herein are methods of treating orpreventing cancer in a subject, comprising modulating levels of one ormore commensal microbes within the subject to: (A) enhance an immuneresponse by the subject, (B) inhibit the growth or spread of the cancer,(C) inhibit immune evasion by the cancer, and/or (D) enhance theefficacy of a therapeutic. In some embodiments, the levels of one ormore commensal microbes are modulated within the gut of the subject. Insome embodiments, modulating the levels of one or more commensalmicrobes comprises increasing and/or decreasing levels of one or morebacterial selected from the genera Adlercreutzia, Oscillopira,Mollicutes, Butyrivibrio, Bacteroides, Clostridium, Fusobacterium,Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus,Bifidobacterium, Rikenella, Alistipes, Marinilabilia, Anaerostipes,Escherichia, and/or Lactobacillus.

In some embodiments, modulating the levels of one or more commensalmicrobes comprises administering a beneficial microbes to the subject.In some embodiments, the beneficial microbes are bacteria. In someembodiments, the bacteria are selected from the genera Adlercreutzia,Oscillopira, Mollicutes, Butyrivibrio, Bacteroides, Clostridium,Fusobacterium, Eubacterium, Ruminococcus, Peptococcus,Peptostreptococcus, Bifidobacterium, Rikenella, Alistipes,Marinilabilia, Anaerostipes, Escherichia, and/or Lactobacillus. In someembodiments, the bacteria are Bifidobacterium. In some embodiments, theBifidobacterium include bacteria selected from the group consisting ofBifidobacterium lactis, Bifidobacterium bifidium, Bifidobacteriumlongum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacteriuminfantis, Bifidobacterium catenulatum, Bifidobacteriumpseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacteriumangulatum. In some embodiments, the beneficial microbes are administeredas a probiotic composition or via microflora transplant from a donor.

In some embodiments, modulating the levels of one or more commensalmicrobes comprises administering one or more antimicrobials. In someembodiments, the antimicrobial kills detrimental microbes. In someembodiments, the antimicrobial is an antibiotic. In some embodiments,methods further comprise administration of beneficial microbes to thesubject.

In some embodiments, methods further comprise administering to thesubject a cancer therapy. In some embodiments, wherein the modulatinglevels of one or more commensal microbes within the subject enhances animmune response by the subject and/or inhibits immune evasion by thecancer, and the cancer therapy is an immunotherapy. In some embodiments,the immunotherapy comprises administration of anti-CTLA-4 antibodiesand/or anti-PD-L1 or anti-PD-1 antibodies. In some embodiments, whereinthe modulating levels of one or more commensal microbes within thesubject enhance the efficacy of a therapeutic, and the cancer therapy issaid therapeutic. In some embodiments, the therapeutic comprises achemotherapeutic. In some embodiments, methods further comprise testingthe subject for immune evasion by the cancer. In some embodiments,methods further comprise surgical, radiation, and/or chemotherapeuticcancer intervention.

In some embodiments, provided herein are kits or compositions comprisinga beneficial commensal microbe and a cancer therapeutic, saidcompositions or components of said kits formulated for therapeuticdelivery to a subject.

In some embodiments, provided herein are beneficial commensal microbesfor use as a medicament in the treatment of cancer and/or inhibition ofimmune evasion.

In some embodiments, provided herein are methods of treating orpreventing cancer in a subject comprising administering to the subjectbacterial formulation comprising bacteria of the genera Bifidobacterium,Rikenella, Alistipes, Marinilabilia, or Anaerostipes. In someembodiments, at least 50% of the bacteria in the bacterial formulationare of the genera Bifidobacterium, Rikenella, Alistipes, Marinilabilia,or Anaerostipes. In some embodiments, at least 90% of the bacteria inthe bacterial formulation are of the genera Bifidobacterium, Rikenella,Alistipes, Marinilabilia, or Anaerostipes. In some embodiments, thebacterial formulation comprise bacteria of the genus Bifidobacterium. Insome embodiments, at least 50% of the bacteria in the bacterialformulation are of the genus Bifidobacterium. In some embodiments, atleast 90% of the bacteria in the bacterial formulation are of the genusBifidobacterium.

In some embodiments, the bacteria of genus Bifidobacterium are selectedfrom the group consisting of Bifidobacterium lactis, Bifidobacteriumbifidium, Bifidobacterium longum, Bifidobacterium animalis,Bifidobacterium breve, Bifidobacterium infantis, Bifidobacteriumcatenulatum, Bifidobacterium pseudocatenulatum, Bifidobacteriumadolescentis, Bifidobacterium angulatum, Bifidobacterium asteroides,Bifidobacterium boum, Bifidobacterium choerinum, Bifidobacteriumcoryneforme, Bifidobacterium cuniculi, Bifidobacterium denticolens,Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacteriumgallinarum, Bifidobacterium indicum, Bifidobacterium inopinatum,Bifidobacterium magnum, Bifidobacterium merycicum, Bifidobacteriumminimum, Bifidobacterium pseudolongum, Bifidobacterium pullorum,Bifidobacterium psychraerophilum, Bifidobacterium ruminantium,Bifidobacterium saeculare, Bifidobacterium scardovii, Bifidobacteriumsimiae, Bifidobacterium subtile, Bifidobacterium therammcidophilum,Bifidobacterium thermophilum, Bifidobacterium tsurumiense,Bifidobacterium urinalis, Bifidobacterium sp.

In some embodiments, the cancer is cancer is selected from the groupconsisting of acute nonlymphocytic leukemia, chronic lymphocyticleukemia, acute granulocytic leukemia, chronic granulocytic leukemia,acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,a leukocythemic leukemia, basophilic leukemia, blast cell leukemia,bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia,Schilling's leukemia, stem cell leukemia, subleukemic leukemia,undifferentiated cell leukemia, hairy-cell leukemia, hemoblasticleukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cellleukemia, acute monocytic leukemia, leukopenic leukemia, lymphaticleukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenousleukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cellleukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocyticleukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinomavillosum, carcinoma gigantocellulare, glandular carcinoma, granulosacell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma,liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoidsarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms'tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma,telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma,multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lungcancer, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, small-cell lung tumors, primary brain tumors, stomachcancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,adrenal cortical cancer, Harding-Passey melanoma, juvenile melanoma,lentigo maligna melanoma, malignant melanoma, acral-lentiginousmelanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman'smelanoma, S91 melanoma, nodular melanoma subungal melanoma, andsuperficial spreading melanoma.

In some embodiments, the subject is human. In some embodiments, thebacterial formulation is administered by oral administration, rectaladministration, topical administration, inhalation or injection. In someembodiments, the bacterial formulation is a food product. In someembodiments, the bacterial formulation comprises at least about 5×10⁶CFU of bacteria. In some embodiments, the bacterial formulation isadministered to the subject in two or more doses. In some embodiments,the administration of at least two of the two or more doses areseparated by at least 1 day. In some embodiments, the administration ofat least two of the two or more doses are separated by at least 1 week.

In some embodiments, methods further comprise administering to thesubject an antibiotic. In some embodiments, the antibiotic isadministered to the subject before the bacterial formulation. In someembodiments, the antibiotic is administered to the subject at least 1day before the bacterial formulation is administered to the subject.

In some embodiments, methods further comprise administering to thesubject an immune checkpoint inhibitor. In some embodiments, the immunecheckpoint inhibitor is a protein or polypeptide that specifically bindsto an immune checkpoint protein. In some embodiments, the immunecheckpoint protein is selected from the group consisting of CTLA4, PD-1,PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. Insome embodiments, the polypeptide or protein is an antibody orantigen-binding fragment thereof. In some embodiments, the immunecheckpoint inhibitor is an interfering nucleic acid molecule. In someembodiments, the interfering nucleic acid molecule is an siRNA molecule,an shRNA molecule or an antisense RNA molecule. In some embodiments, theimmune checkpoint inhibitor is selected from the group consisting ofnivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110,TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT 011, MPDL3280A,MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010. In some embodiments, theimmune checkpoint inhibitor is administered before the bacterialformulation. In some embodiments, the immune checkpoint inhibitor isadministered at least one day before the bacterial formulation. In someembodiments, the immune checkpoint is administered at about the sametime as the bacterial formulation. In some embodiments, the immunecheckpoint inhibitor is administered on the same day as the bacterialformulation. In some embodiments, the immune checkpoint inhibitor isadministered after the bacterial formulation. In some embodiments, theimmune checkpoint inhibitor is administered at least one day after thebacterial formulation. In some embodiments, the immune checkpointinhibitor is administered by injection. In some embodiments, theinjection is an intravenous, intramuscular, intratumoral or subcutaneousinjection.

In some embodiments, provided herein are methods of treating cancer in ahuman subject comprising administering to the subject an immunecheckpoint inhibitor and a bacterial formulation comprising bacteria ofthe genera Bifidobacterium. In some embodiments, at least 50% (e.g.,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, orranges therebetween) of the bacteria in the bacterial formulation are ofthe genera Bifidobacterium. In some embodiments, at least 90% (e.g.,90%, 95%, 99%, 99.9%, 99.99%, or more or ranges therebetween) of thebacteria in the bacterial formulation are of the genera Bifidobacterium.In some embodiments, the bacteria of the genus Bifidobacterium comprisebacteria of the species Bifidobacterium lactis, Bifidobacteriumbifidium, Bifidobacterium longum, Bifidobacterium animalis,Bifidobacterium breve, Bifidobacterium infantis, Bifidobacteriumcatenulatum, Bifidobacterium pseudocatenulatum, Bifidobacteriumadolescentis, Bifidobacterium angulatum, Bifidobacterium asteroides,Bifidobacterium boum, Bifidobacterium choerinum, Bifidobacteriumcoryneforme, Bifidobacterium cuniculi, Bifidobacterium denticolens,Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacteriumgallinarum, Bifidobacterium indicum, Bifidobacterium inopinatum,Bifidobacterium magnum, Bifidobacterium merycicum, Bifidobacteriumminimum, Bifidobacterium pseudolongum, Bifidobacterium pullorum,Bifidobacterium psychraerophilum, Bifidobacterium ruminantium,Bifidobacterium saeculare, Bifidobacterium scardovii, Bifidobacteriumsimiae, Bifidobacterium subtile, Bifidobacterium therammcidophilum,Bifidobacterium thermophilum, Bifidobacterium tsurumiense,Bifidobacterium urinalis or Bifidobacterium sp. In some embodiments, thebacterial formulation is administered by oral administration or rectaladministration. In some embodiments, the bacterial formulation isadministered by oral administration. In some embodiments, the bacterialformulation comprises at least 5×10⁶ CFU (e.g., 5×10⁶ CFU, 1×10⁷ CFU,2×10⁷ CFU, 5×10⁷ CFU, 1×10⁸ CFU, 2×10⁸ CFU, 5×10⁸ CFU, 1×10⁹ CFU, 2×10⁹CFU, 5×10⁹ CFU, 1×10¹⁰ CFU, 2×10¹⁰ CFU, 5×10¹⁰ CFU, 1×10¹¹ CFU, 2×10¹¹CFU, 5×10¹¹ CFU, 1×10¹² CFU, 2×10¹² CFU, 5×10¹² CFU, or more or rangestherebetween) of bacteria of the genera Bifidobacterium. In someembodiments, the bacterial formulation is administered to the subject intwo or more doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or rangestherebetween). In some embodiments, the administration of doses areseparated by at least 1 week. In some embodiments, methods furthercomprise administering to the subject an antibiotic prior to theadministration of the bacterial formulation. In some embodiments, theantibiotic is administered to the subject at least 1 day before thebacterial formulation is administered to the subject. In someembodiments, the immune checkpoint inhibitor is a protein or polypeptidethat binds to an immune checkpoint protein. In some embodiments, theimmune checkpoint inhibitor is an antibody or antigen binding fragmentthereof that binds to an immune checkpoint protein. In some embodiments,the immune checkpoint protein is CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3,B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. In some embodiments, the immunecheckpoint protein is PD-1 or PD-L1. In some embodiments, the immunecheckpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, AMP-224,AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475,CT 011, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010. Insome embodiments, the immune checkpoint inhibitor is administered byintravenous injection, intramuscular injection, intratumoral injectionor subcutaneous injection.

In some embodiments, provided herein are methods of treating cancer in ahuman subject comprising administering to the subject a bacterialformulation comprising at least 5×10⁶ CFU (e.g., 5×10⁶ CFU, 1×10⁷ CFU,2×10⁷ CFU, 5×10⁷ CFU, 1×10⁸ CFU, 2×10⁸ CFU, 5×10⁸ CFU, 1×10⁹ CFU, 2×10⁹CFU, 5×10⁹ CFU, 1×10¹⁰ CFU, 2×10¹⁰ CFU, 5×10¹⁰ CFU, 1×10¹¹ CFU, 2×10¹¹CFU, 5×10¹¹ CFU, 1×10¹² CFU, 2×10¹² CFU, 5×10¹² CFU, or more or rangestherebetween) of bacteria of the genera Bifidobacterium, wherein atleast 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%,or more, or ranges therebetween) of the bacteria in the bacterialformulation are of the genera Bifidobacterium. In some embodiments, atleast 90% (e.g., 90%, 95%, 99%, 99.9%, 99.99%, or more or rangestherebetween) of the bacteria in the bacterial formulation are of thegenera Bifidobacterium. In some embodiments, the bacteria of the genusBifidobacterium comprise bacteria of the species Bifidobacterium lactis,Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacteriumanimal's, Bifidobacterium breve, Bifidobacterium infant's,Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum,Bifidobacterium adolescent's, Bifidobacterium angulatum, Bifidobacteriumasteroides, Bifidobacterium boum, Bifidobacterium choerinum,Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacteriumdenticolens, Bifidobacterium dentium, Bifidobacterium gallicum,Bifidobacterium gallinarum, Bifidobacterium indicum, Bifidobacteriuminopinatum, Bifidobacterium magnum, Bifidobacterium merycicum,Bifidobacterium minimum, Bifidobacterium pseudolongum, Bifidobacteriumpullorum, Bifidobacterium psychraerophilum, Bifidobacterium ruminantium,Bifidobacterium saeculare, Bifidobacterium scardovii, Bifidobacteriumsimiae, Bifidobacterium subtile, Bifidobacterium therammcidophilum,Bifidobacterium thermophilum, Bifidobacterium tsurumiense,Bifidobacterium urinal's or Bifidobacterium sp. In some embodiments, thebacterial formulation is administered by oral administration or rectaladministration. In some embodiments, the bacterial formulation isadministered by oral administration. In some embodiments, the bacterialformulation is administered to the subject in two or more doses (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or ranges therebetween). In someembodiments, methods further comprise administering to the subject anantibiotic before the bacterial formulation is administered to thesubject. In some embodiments, methods further comprise administering tothe subject an immune checkpoint inhibitor. In some embodiments, theimmune checkpoint inhibitor is an antibody or antigen binding fragmentthereof that binds to CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4,BTLA, KIR, LAG3, TIM-3 or VISTA. In some embodiments, the immunecheckpoint inhibitor is an antibody or antigen binding fragment thereofthat binds to PD-1 or PD-L1. In some embodiments, the immune checkpointinhibitor is nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514,STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT 011,MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-H. Differences in melanoma outgrowth and tumor-specific immuneresponses between C57BL/6 JAX and TAC mice are eliminated uponcohousing. (A) B16.SIY tumor growth kinetics in newly arrived JAX andTAC mice. (B) IFN-γ ELISPOT in tumor-bearing JAX and TAC mice 7 daysfollowing tumor inoculation. (C) Mean size of IFN-γ spots (10⁻³ mm²).(D) Percentage of STY⁺ T cells of total CD8⁺ T cells within the tumor ofJAX and TAC mice as determined by flow cytometry 21 days post-tumorinoculation. Representative plots (left), quantification (right). (E)B16.SIY tumor growth kinetics in JAX and TAC mice cohoused for 3 weeksprior to tumor inoculation. (F) Number of IFN-γ spots/10⁶ splenocytes intumor-bearing JAX and TAC mice cohoused for 3 weeks prior to tumorinoculation. (G) Mean size of IFN-γ spots (10⁻³ mm²). (H) Percentage ofSTY⁺ T cells of total CD8⁺ T cells within the tumor of JAX and TAC micecohoused for 3 weeks prior to tumor inoculation.

FIG. 2A-G. Oral administration of JAX fecal material to TAC miceenhances spontaneous anti-tumor immunity and response to αPD-L1 mAbtherapy. (A) B16.SIY tumor growth in newly arrived TAC mice, TAC and JAXmice orally gavaged with PBS, TAC or JAX fecal material prior to tumorimplantation. (B) Number of IFN-γ spots x mean spot size (10⁻³ mm²),determined by ELISPOT 7 days following tumor inoculation. (C) Percentageof STY⁺ CD8⁺ T cells within the tumor of TAC and JAX mice treated as in(A), 21 days post-tumor inoculation. Representative plots (left),quantification (right). (D) B16.SIY tumor growth in TAC mice, untreatedor treated with JAX fecal material 7 and 14 days post tumorimplantation, αPD-L1 mAb 7, 10, 13 and 16 days post tumor implantation,or both regimens. (E) IFN-γ ELISPOT assessed 5 days after start oftreatment. (F) Percentage of tumor-infiltrating STY⁺ CD8⁺ T cells,determined by flow cytometry 14 days after start of treatment. (G)B16.SIY tumor growth kinetics in TAC and JAX mice, untreated or treatedwith αPD-L1 mAb 7, 10, 13 and 16 days post tumor implantation.

FIG. 3A-G. Direct administration of Bifidobacterium to TAC recipientswith established tumors improves tumor-specific immunity and response toαPD-L1 mAb therapy. (A-C) Bacterial species diversity (A), principalcoordinate analysis plot of bacterial β-diversity (B) and operationaltaxonomic unit (OTU) levels of top Bifidobacterium taxon (C) in fecalmaterial obtained from JAX, TAC, TAC-fed TAC and JAX-fed TAC mice.Comparisons in A-C were performed using 9-10 replicates from each vendorand 4-5 replicates from each treatment. (D) B16.SIY tumor growthkinetics in TAC mice, untreated or treated with Bifidobacterium 7 and 14days post tumor implantation (white arrows), αPD-L1 mAb 7, 10, 13 and 16days post tumor implantation (black arrows) or both regimens. (E) IFN-γELISPOT assessed 5 days after start of treatment. (F) Percentage oftumor-infiltrating STY⁺ CD8⁺ T cells, determined by flow cytometry 14days following start of treatment. Representative plots (left),quantification of data combined from 2 independent experiments (right).(G) B16.SIY tumor growth for isotype-treated (left) or CD8-depleted(right) groups as in D.

FIG. 4A-E. Dendritic cells isolated from JAX and Bifidobacterium-fed TACmice show increased expression of genes associated with antitumorimmunity and heightened capability for T cell activation (A)Quantification of IFN-γ MFI (mean fluorescence intensity) of 2C CD8⁺ Tcells in the tumor-draining lymph node (left) and spleen (right) of TAC,JAX, Bifidobacterium-fed TAC mice on day 7 post adoptive transfer. (B)Percentage of MHC Class IIhi DCs in tumors isolated from TAC, JAX, andBifidobacterium-fed TAC mice 40 hours post tumor implantation asassessed by flow cytometry. (C) Enriched biological pathways andfunctions found within the subset of elevated genes in JAX andBifidobacterium-treated TAC-derived DCs relative to untreated TAC DCsisolated from tumors 40 hrs post tumor inoculation, as assessed by DAVIDpathway analysis. Bars indicate the percent of genes in a pathwayupregulated in DCs isolated from JAX and Bifidobacterium-fed TAC mice.Line indicates p-values calculated by Fisher's exact test. (D) Heat mapof key antitumor immunity genes in DCs isolated from JAX,Bifidobacterium-treated TAC or untreated TAC mice. Mean fold-change foreach gene transcript is shown on the right. (E) Quantification of IFN-γ⁺2C TCR Tg CD8⁺ T cells stimulated in vitro with DCs purified fromperipheral lymphoid tissues of naïve TAC, JAX, andBifidobacterium-treated TAC mice in the presence of differentconcentrations of SIY peptide.

FIG. 5A-D. (A) Schematic of prophylactic fecal transfer: fecal pelletscollected from JAX and TAC mice upon arrival in our facility wereresuspended in PBS, homogenized and the supernatant was introduced byoral gavage into either JAX or TAC recipients as shown, once a week fortwo weeks prior to B16.SIY tumor inoculation. (B) B16.SIY tumor growthin JAX mice orally gavaged with TAC or JAX fecal material once weeklyfor two weeks prior to tumor implantation. (C) Percentage of STY⁺ Tcells of total CD8⁺ T cells within the tumor of groups as in FIG. 2A,determined by flow cytometry 7 days post-tumor inoculation. (D)Percentage of STY⁺ T cells of total CD8⁺ T cells within the tumor of JAXand TAC mice, untreated or treated with αPD-L1 mAb, as determined byflow cytometry 21 days post-tumor inoculation.

FIG. 6A-H. (A) Relative abundance of all taxa combined belonging to theBifidobacterium genus in fecal material obtained from TAC, JAX, TAC-fedTAC and JAX-fed TAC mice. Comparisons were performed using 9-10replicates from each vendor and 4-5 replicates from each treatment. (B)Number of colony forming units (CFU) of live and heat inactivatedbifidobacteria, plated in RCM agar following serial dilution in reducedPBS and incubated in an anaerobic chamber for 72 hours. (C) B16.SIYtumor growth kinetics in TAC mice, untreated or treated with liveBifidobacterium, heat inactivated Bifidobacterium or JAX fecal material7 and 14 days post tumor implantation. (D) Percentage oftumor-infiltrating SIY⁺ T cells of total CD8⁺ T cells for treatmentgroups as in C, determined by flow cytometry 14 days after start oftreatment. C-D show data combined from 2-4 independent experiments, 5mice per group. (E) B16.F10 tumor growth kinetics in TAC mice, untreatedor treated with Bifidobacterium 7 and 14 days post tumor implantation.(F) MB49 tumor growth kinetics in TAC mice, untreated or treated withBifidobacterium 7 and 14 days post tumor implantation. (G) B16.SIY tumorgrowth kinetics in TAC mice, untreated or treated with Lactobacillusmurinus or JAX fecal material 7 and 14 days post tumor implantation. (H)Percentage of tumor-infiltrating STY⁺ T cells of total CD8⁺ T cells fortreatment groups as in G, determined by flow cytometry 18 days afterstart of treatment.

FIG. 7A-B. (A) Schematic of in vivo 2C proliferation assays. CD8+ Tcells were isolated from the spleen and lymph node of naïve 2C TCR TgCD45.1+/.2+mice, labeled with CFSE and injected i.v. into CD45.2+C57BL/6mice derived from either TAC, JAX or Bifidobacterium-treated TAC mice.24 hours later, mice were inoculated with 1×106 B16.SIY melanoma cellss.c. Spleen and tumor-draining lymph node were harvested andrestimulated ex-vivo with SIY peptide. Intracellular IFN-γ productionand CFSE dilution were assessed in gated CD45.1+/.2+2C T cells by flowcytometry; TDLN=tumordraining lymph node. (B) Representative CFSEdilution assessed in gated CD45.1+/.2+2C T cells by flow cytometry(left) and quantification (right).

FIG. 8A-G. Direct administration of Bifidobacterium to TAC recipientswith established tumors improves tumor-specific immunity and response toαPD-L1 mAb therapy. (A) Principal coordinate analysis plot of bacterialβ-diversity over time in groups treated as in FIG. 2A. (B) Phylogeneticanalysis of taxa that are of significantly different abundance in newlyarrived JAX vs TAC mice FDR<0.05 (non-parametric t test); bars representlog-transformed fold changes, inner circle=log 10(10); middle circle=log10(100); outer circle=log 10(1000). (C) Heatmap demonstrating relativeabundance over time of significantly altered genus-level taxa in JAX-fedTAC mice FDR<0.05 (non-parametric t test); columns depict individualmice; each timepoint shows mice from two separate cages, 3-4 mice percage. (D) Correlation plot of relative abundance of Bifidobacterium OTU681370 in fecal material obtained from groups as in (A) 14 days postarrival and frequency of STY⁺ CD8⁺ T cells in tumor; p=1.4×10-5,FDR=0.0002, R2=0.86 (univariate regression). (E) B16.SIY tumor growthkinetics in TAC mice, untreated or treated with Bifidobacterium 7 and 14days post tumor implantation, αPD-L1 mAb 7, 10, 13 and 16 days posttumor implantation, or both regimens. (F) IFN-γ ELISPOT assessed 5 daysafter start of treatment. (G) Percentage of tumor-infiltrating STY⁺ CD8⁺T cells, determined by flow cytometry 14 days following start oftreatment.

FIG. 9A-E. (A) Relative abundance of Bifidobacterium OTU_681370 in fecalmaterial obtained from TAC mice 7 days following inoculation withcommercial Bifidobacterium species. (B) Bifidobacterium levels in fecalmaterial obtained from groups as shown, assessed by qPCR usinggenus-specific primers. (C) Representative plots showing percentage ofSTY⁺ T cells of total CD8⁺ T cells within the tumor of untreated andBifidobacterium-treated TAC mice, as assessed by flow cytometry 14 daysfollowing start of treatment. (D) Bifidobacterium levels in TAC mice 3weeks post Bifidobacterium administration, assessed by qPCR. (E) B16.SIYtumor growth in TAC mice, untreated or inoculated with Bifidobacterium 6weeks prior to tumor implantation.

FIG. 10A-D. (A) B16.SIY tumor growth for isotype-treated (left) orCD8-depleted (right) groups as in FIG. 3E. (B) Number of colony formingunits (CFU) of live and heat inactivated bifidobacteria, plated in RCMagar following serial dilution in reduced PBS and incubated in ananaerobic chamber for 72 hours. Bars represent 2 replicate plates ofeach dilution. (C) B16.SIY tumor growth kinetics in TAC mice, untreatedor treated with live Bifidobacterium, heat inactivated Bifidobacteriumor JAX fecal material 7 and 14 days post tumor implantation. (D)Percentage of tumor-infiltrating STY⁺ T cells of total CD8⁺ T cells fortreatment groups as in (C), determined by flow cytometry 14 days afterstart of treatment.

FIG. 11A-E. (A) B16.SIY tumor growth kinetics in TAC mice, untreated ortreated with ATCC derived B. breve or B. longum. (B) B16.F10 tumorgrowth kinetics in TAC mice, untreated or treated with Bifidobacterium 7and 14 days post tumor implantation. (C) MB49 tumor growth kinetics inTAC mice, untreated or treated with Bifidobacterium 7 and 14 days posttumor implantation. (D) B16.SIY tumor growth kinetics in TAC mice,untreated or treated with Lactobacillus murinus or JAX fecal material 7and 14 days post tumor implantation. (E) Percentage oftumor-infiltrating STY⁺ T cells of total CD8⁺ T cells for treatmentgroups as in (D), determined by flow cytometry 18 days after start oftreatment.

FIG. 12A-C. (A) Heatmap demonstrating relative abundance ofsignificantly altered genus-level taxa in Bifidobacterium-fed TAC miceFDR<0.05 (non-parametric t-test); columns depict individual mice; n=4-8mice per group. (B) Frequency of CD4+ FOXP3+ T cells in tumors isolatedfrom JAX and TAC mice 21 days post tumor inoculation, assessed by flowcytometry; representative plot (top), quantification (bottom). (C)Evaluation of translocation of Bifidobacterium into mesenteric lymphnodes (mLN), spleen and tumor of TAC, JAX and Bifidobacterium-inoculatedmice, assessed by qPCR.

FIG. 13A-C. (A) Representative plots depicting the strategy forisolation of DCs from tumors in JAX, TAC and Bifidobacterium-treated TACmice: live CD45+CD3-CD19-MHCIIhiCD11c+ dendritic cells were sorted asshown. (B) All enriched biological pathways and functions found withinthe subset of elevated genes (fold change≥1.5) in JAX andBifidobacterium-treated TAC-derived DCs relative to untreated TAC DCsisolated from tumors 40 hrs post inoculation, as assessed by DAVIDpathway analysis. (C) qPCR validation of genes identified by microarraygene expression profiling as in (B).

FIG. 14A-B. (A) Representative flow plots of CFSE dilution and IFN-γproduction in 2C CD8+ T cells stimulated in vitro with DCs purified fromnaive TAC, JAX and Bifidobacterium-treated TAC mice in the presence ofdifferent concentrations of SIY peptide as shown. (B) Percentage of 2CCD8+ T cells with undiluted CFSE, stimulated in vitro with DCs purifiedfrom naive TAC, JAX and Bifidobacterium-treated TAC mice in the presenceof different concentrations of SIY peptide as shown.

FIG. 15. B16.SIY tumor growth in TAC mice, untreated or treatedindividually with ATCC 15700 B. breve, ATCC BAA-999 B. longum, ATCC27536 B. Lactis or ATCC 15696 B. Bifidum, or treated with all fourstrains combined.

Definitions

As used herein, the term “microbe” refers to cellular microorganismsincluding bacteria, fungi, and archaea, and encompasses both individualorganisms and populations comprising any number of the organisms.

As used herein, the term “microflora” refers to an assemblage ofmicroorganisms localized to a distinct environment. Microflora mayinclude, for example, populations of various bacteria, fungi, and/orarchaea that inhabit a particular environment. For example, “gutmicroflora,” “vaginal microbiota,” and “oral microflora” are anassemblage of one or more species of microorganisms that are localizedto, or found in, the gut, vagina, or mouth, respectively. “Normalmicroflora” refers to a population of microorganisms that localize in aparticular environment in a normal, non-pathological state (e.g., asample of gut microflora from a subject without cancer). “Pathologicmicroflora” refers to a population of various microorganisms thatlocalize in a particular environment in pathological state and differsfrom normal microflora in terms of identify, absolute amount, orrelative amount of the various microbes.

As used herein, the term “commensal microbe” refers to a microorganismthat is non-pathogenic to a host and is part of the normal microflora ofthe host.

As used herein, the term “co-administration” refers to theadministration of at least two agents (e.g., commensal microflora and acancer therapy) or therapies to a subject. In some embodiments, theco-administration of two or more agents/therapies is concurrent. Inother embodiments, the co-administration of two or more agents/therapiesis sequential (e.g., a first agent/therapy is administered prior to asecond agent/therapy).

As used herein, the term “beneficial microbe” refers to a microbe (e.g.,bacterium) strain or species that inhibits the growth of cancer/tumorcells and/or facilitates treatment of cancer/tumor cells (e.g., inhibitsimmune evasion). Beneficial microbes may function by, for example,creating an anti-cancer/anti-tumor environment, microenvironment and/ormetabolome, and/or by creating an environment, microenvironment and/ormetabolome that inhibits immune evasion or other mechanisms by whichcancer cells resist therapy.

As used herein, the term “detrimental microbe” refers to a microbe(e.g., bacterium) strain or species that facilitates the growth ofcancer/tumor cells and/or prevents or reduces the effectiveness oftreatment of cancer/tumor cells (e.g., inhibits immune evasion).Detrimental microbes may function by, for example, creating anenvironment, microenvironment and/or metabolome that facilitates immuneevasion or other mechanisms by which cancer cells resist therapy and/orenhance cancer/tumor growth.

As used herein, the term “pharmaceutical agent” refers to a compound,macromolecule, or other chemical/non-biological entity that isadministered to a subject to elicit a desired biological response. Apharmaceutical agent may be a “drug” or another entity which isbiologically active in a human being or other mammal, locally and/orsystemically. Examples of drugs are disclosed in the Merck Index and thePhysicians Desk Reference, the entire disclosures of which areincorporated by reference herein for all purposes.

As used herein, the terms “microbial agent,” “commensal microbialagent,” and “probiotic” refer to compositions comprising a microbe orpopulation of multiple different microbes for administration to asubject.

As used herein, the term “antimicrobial agent” is used to describe atherapeutic compound or bioactive agent which treats a microbialinfection, for example, an infection caused by a bacteria, virus,protozoa or fungus. The antimicrobial agent may be an antibiotic, anantifungal agent, an antiviral or an antiprotozoal or antiparasiticagent (which may also be used to treat multicellular parasites).

As used herein, the terms “antibiotic” and “antibacterial agent” referto a chemical agent which is active against bacteria. In common usage,an antibiotic is a substance or compound (also called chemotherapeuticagent) that kills or inhibits the growth of bacteria. Antibacterialantibiotics can be categorized based on their target specificity:“narrow-spectrum” antibiotics target particular types of bacteria, suchas Gram-negative or Gram-positive bacteria, while broad-spectrumantibiotics affect a wide range of bacteria. Antibiotics which targetthe bacterial cell wall (e.g., penicillins, cephalosporins, cephems), orcell membrane (e.g., polymixins), or interfere with essential bacterialenzymes (e.g., quinolones, sulfonamides) usually are bactericidal innature. Those which target protein synthesis such as theaminoglycosides, macrolides and tetracyclines are usuallybacteriostatic. Three newer classes of antibiotics include: cycliclipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), andoxazolidinones (e.g., linezolid). Tigecycline is a broad-spectrumantibiotic, while the two others are useful for Gram-positiveinfections.

As used herein, the term “antiviral agent” refers to a chemical agentwhich is used to treat a viral infection. Antiviral drugs are a class ofmedication used specifically for treating viral infections, specificantivirals are useful for treating infection by specific viruses.Antivirals typically only inhibit virus development.

As used herein, the term “antifungal agent” refers to a therapeuticcompound or bioactive agent which may be used to treat a fungalinfection in a patient. An antifungal drug is a medication used to treatfungal infections such as athlete's foot, ringworm, candidiasis(thrush), serious systemic infections such as cryptococcal meningitis,and related fungal infections. Antifungal agents include, for example,polyene antifungals, imidazole, triazole and thiazole antifungals,allylamines, echinocandins, griseofulvin, flycystosine, undecylenicacid, among others.

As used herein, the term “antiparasitic agent” refers to a therapeuticcompound or bioactive agent that is used to treat parasitic diseasesincluding nematodes, cestodes, trematodes, infectious protozoa, andamoebas. Exemplary antiparasitic agents include: antinematodes (e.g.,mebendazole, pyrantel pamoate, thiabendazole, diethycarbazine),anticestodes (e.g., niclosamide, praziquantel), antitrematodes (e.g.,praziquantel), antiamoebics (e.g., rifampin and amphotericin B),antiprotozoals (e.g., melarsoprol, eflornithine, metronidazole andtinidazole), among others.

As used herein, the term “pharmaceutical formulation” refers to at leastone pharmaceutical agent and/or microbial agent in combination with oneor more additional components that assist in rendering the agent(s)suitable for achieving the desired effect upon administration to asubject. The pharmaceutical formulation may include one or moreadditives, for example pharmaceutically acceptable excipients, carriers,penetration enhancers, coatings, stabilizers, buffers or other materialsphysically associated with the pharmaceutical/microbial agent to enhancethe administration, release (e.g., timing of release), deliverability,bioavailability, effectiveness, etc. of the dosage form. The formulationmay be, for example, a liquid, a suspension, a solid, a nanoparticle,emulsion, micelle, ointment, gel, emulsion, coating, etc. Apharmaceutical formulation may contain a single agent or multiple agents(e.g., microbial agent and pharmaceutical agent).

As used herein, the term “subject” broadly refers to any animal,including but not limited to, human and non-human animals (e.g., dogs,cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As usedherein, the term “patient” typically refers to a subject that is beingtreated for a disease or condition (e.g., cancer, solid tumor cancer,non-T cell-infiltrated tumor cancer, etc.).

As used herein, an “immune response” refers to the action of a cell ofthe immune system (e.g., T lymphocytes, B lymphocytes, natural killer(NK) cells, macrophages, eosinophils, mast cells, dendritic cells,neutrophils, etc.) and soluble macromolecules produced by any of thesecells or the liver (including Abs, cytokines, and complement) thatresults in selective targeting, binding to, damage to, destruction of,and/or elimination from a subject of invading pathogens, cells ortissues infected with pathogens, or cancerous or other abnormal cells.

As used herein, the term “immunoregulator” refers to an agent or asignaling pathway (or a component thereof) that regulates an immuneresponse. “Regulating,” “modifying” or “modulating” an immune responserefers to any alteration of the immune system or in the activity of suchcell. Such regulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunoregulators have been identified,some of which may have enhanced function in a cancer microenvironment.

As used herein, the term “immune evasion” refers to inhibition of asubject's immune system or a component thereof (e.g., endogenous T cellresponse) by a cancer or tumor cell in order to maximize or allowcontinued growth or spread of the cancer/tumor.

As used herein, the term “immunotherapy” refers to the treatment orprevention of a disease or condition (e.g., cancer) by a methodcomprising inducing, enhancing, suppressing or otherwise modifying animmune response.

As used herein, “potentiating an endogenous immune response” meansincreasing the effectiveness or potency of an existing immune responsein a subject. This increase in effectiveness and potency may beachieved, for example, by overcoming mechanisms that suppress theendogenous host immune response or by stimulating mechanisms thatenhance the endogenous host immune response.

As used herein, the term “antibody” refers to a whole antibody moleculeor a fragment thereof (e.g., fragments such as Fab, Fab′, and F(ab′)2),it may be a polyclonal or monoclonal antibody, a chimeric antibody, ahumanized antibody, a human antibody, etc.

A native antibody typically has a tetrameric structure. A tetramertypically comprises two identical pairs of polypeptide chains, each pairhaving one light chain (in certain embodiments, about 25 kDa) and oneheavy chain (in certain embodiments, about 50-70 kDa). In a nativeantibody, a heavy chain comprises a variable region, VH, and threeconstant regions, CH1, CH2, and CH3. The VH domain is at theamino-terminus of the heavy chain, and the CH3 domain is at thecarboxy-terminus. In a native antibody, a light chain comprises avariable region, VL, and a constant region, CL. The variable region ofthe light chain is at the amino-terminus of the light chain. In a nativeantibody, the variable regions of each light/heavy chain pair typicallyform the antigen binding site. The constant regions are typicallyresponsible for effector function.

In a native antibody, the variable regions typically exhibit the samegeneral structure in which relatively conserved framework regions (FRs)are joined by three hypervariable regions, also called complementaritydetermining regions (CDRs). The CDRs from the two chains of each pairtypically are aligned by the framework regions, which may enable bindingto a specific epitope. From N-terminus to C-terminus, both light andheavy chain variable regions typically comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The CDRs on the heavy chain are referredto as H1, H2, and H3, while the CDRs on the light chain are referred toas L1, L2, and L3. Typically, CDR3 is the greatest source of moleculardiversity within the antigen-binding site. H3, for example, in certaininstances, can be as short as two amino acid residues or greater than26. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat et al. (1991) Sequences ofProteins of Immunological Interest (National Institutes of Health,Publication No. 91-3242, vols. 1-3, Bethesda, Md.); Chothia, C., andLesk, A. M. (1987) J. Mol. Biol. 196:901-917; or Chothia, C. et al.Nature 342:878-883 (1989). In the present application, the term “CDR”refers to a CDR from either the light or heavy chain, unless otherwisespecified.

As used herein, the term “heavy chain” refers to a polypeptidecomprising sufficient heavy chain variable region sequence to conferantigen specificity either alone or in combination with a light chain.

As used herein, the term “light chain” refers to a polypeptidecomprising sufficient light chain variable region sequence to conferantigen specificity either alone or in combination with a heavy chain.

As used herein, when an antibody or other entity “specificallyrecognizes” or “specifically binds” an antigen or epitope, itpreferentially recognizes the antigen in a complex mixture of proteinsand/or macromolecules, and binds the antigen or epitope with affinitywhich is substantially higher than to other entities not displaying theantigen or epitope. In this regard, “affinity which is substantiallyhigher” means affinity that is high enough to enable detection of anantigen or epitope which is distinguished from entities using a desiredassay or measurement apparatus. Typically, it means binding affinityhaving a binding constant (Ka) of at least 10⁷M⁻¹ (e.g., >10⁷M⁻¹,>10⁸M⁻¹, >10⁹ M⁻¹, >10¹⁰ M⁻¹, >10¹¹M⁻¹, >10¹²M⁻¹, >10¹³ M⁻¹, etc.). Incertain such embodiments, an antibody is capable of binding differentantigens so long as the different antigens comprise that particularepitope. In certain instances, for example, homologous proteins fromdifferent species may comprise the same epitope.

As used herein, the term “monoclonal antibody” refers to an antibodywhich is a member of a substantially homogeneous population ofantibodies that specifically bind to the same epitope. In certainembodiments, a monoclonal antibody is secreted by a hybridoma. Incertain such embodiments, a hybridoma is produced according to certainmethods known to those skilled in the art. See, e.g., Kohler andMilstein (1975) Nature 256: 495-499; herein incorporated by reference inits entirety. In certain embodiments, a monoclonal antibody is producedusing recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Incertain embodiments, a monoclonal antibody refers to an antibodyfragment isolated from a phage display library. See, e.g., Clackson etal. (1991) Nature 352: 624-628; and Marks et al. (1991) J. Mol. Biol.222: 581-597; herein incorporated by reference in their entireties. Themodifying word “monoclonal” indicates properties of antibodies obtainedfrom a substantially-homogeneous population of antibodies, and does notlimit a method of producing antibodies to a specific method. For variousother monoclonal antibody production techniques, see, e.g., Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.); herein incorporated by referencein its entirety.

As used herein, the term “antibody fragment” refers to a portion of afull-length antibody, including at least a portion antigen bindingregion or a variable region. Antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, diabodies, and otherantibody fragments that retain at least a portion of the variable regionof an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med.9:129-134; herein incorporated by reference in its entirety. In certainembodiments, antibody fragments are produced by enzymatic or chemicalcleavage of intact antibodies (e.g., papain digestion and pepsindigestion of antibody) produced by recombinant DNA techniques, orchemical polypeptide synthesis.

For example, a “Fab” fragment comprises one light chain and the CH1 andvariable region of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule. A “Fab”′fragment comprises one light chain and one heavy chain that comprisesadditional constant region, extending between the CH1 and CH2 domains.An interchain disulfide bond can be formed between two heavy chains of aFab′ fragment to form a “F(ab′)2” molecule.

An “Fv” fragment comprises the variable regions from both the heavy andlight chains, but lacks the constant regions. A single-chain Fv (scFv)fragment comprises heavy and light chain variable regions connected by aflexible linker to form a single polypeptide chain with anantigen-binding region. Exemplary single chain antibodies are discussedin detail in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203;herein incorporated by reference in their entireties. In certaininstances, a single variable region (e.g., a heavy chain variable regionor a light chain variable region) may have the ability to recognize andbind antigen.

Other antibody fragments will be understood by skilled artisans.

As used herein, the term “chimeric antibody” refers to an antibody madeup of components from at least two different sources. In certainembodiments, a chimeric antibody comprises a portion of an antibodyderived from a first species fused to another molecule, e.g., a portionof an antibody derived from a second species. In certain suchembodiments, a chimeric antibody comprises a portion of an antibodyderived from a non-human animal fused to a portion of an antibodyderived from a human. In certain such embodiments, a chimeric antibodycomprises all or a portion of a variable region of an antibody derivedfrom a non-human animal fused to a constant region of an antibodyderived from a human.

A “humanized” antibody refers to a non-human antibody that has beenmodified so that it more closely matches (in amino acid sequence) ahuman antibody. A humanized antibody is thus a type of chimericantibody. In certain embodiments, amino acid residues outside of theantigen binding residues of the variable region of the non-humanantibody are modified. In certain embodiments, a humanized antibody isconstructed by replacing all or a portion of a complementaritydetermining region (CDR) of a human antibody with all or a portion of aCDR from another antibody, such as a non-human antibody, having thedesired antigen binding specificity. In certain embodiments, a humanizedantibody comprises variable regions in which all or substantially all ofthe CDRs correspond to CDRs of a non-human antibody and all orsubstantially all of the framework regions (FRs) correspond to FRs of ahuman antibody. In certain such embodiments, a humanized antibodyfurther comprises a constant region (Fc) of a human antibody.

The term “effective dose” or “effective amount” refers to an amount ofan agent, e.g., an antibody, that results in the reduction of symptomsin a patient or results in a desired biological outcome. In certainembodiments, an effective dose or effective amount is sufficient totreat or reduce symptoms of a disease or condition.

DETAILED DESCRIPTION

Provided herein are methods of treatment and/or prevention of cancer bymanipulation of commensal microflora. In particular, the amount,identity, presence, and/or ratio of microflora (e.g., gut microflora) ina subject is manipulated to facilitate one or more co-treatments.

T cell infiltration of solid tumors is associated with favorable patientoutcomes, yet the mechanisms underlying variable endogenous immuneresponses between individuals are not well understood. Experiments wereconducted during development of embodiments described herein to examinepotential effects of microbial composition on spontaneous anti-tumorimmunity. B16 melanoma growth was compared in C57BL/6 mice havingdistinct commensal microbiota. The two populations of mice showed robustversus weak spontaneous anti-tumor immunity. This phenotypic differencewas eliminated upon cohousing or following fecal transfer. 16S rRNAsequencing identified Bifidobacterium as associated with the anti-tumoreffects. Oral administration of Bifidobacterium alone or in combinationwith systemic αPD-L1 in tumor-bearing mice markedly improved tumorcontrol in a CD8⁺ T cell-dependent manner. Mechanistically, the effectwas mediated by augmented dendritic cell function leading to more robustantigen-specific CD8⁺ T cell priming and markedly increased accumulationof activated T cells in the tumor microenvironment. These data, forexample, demonstrate advantages manipulating commensal microbes as acancer therapeutic.

In some embodiments, the effectiveness of an endogenous immune response,immunotherapy, chemotherapeutic, or other treatment (e.g., surgery,radiation, etc.) in the treatment or prevention of reoccurrence ofcancer and/or tumor is dependent upon conditions within the subject(e.g., the tumor microenvironment). In particular, the identity orcharacteristics (e.g., concentration or level) of the microflora withina subject affects the effectiveness of cancer treatments (e.g.,generally or specific treatments) and/or the effectiveness of thesubject's own immune response to cancer.

In some embodiments, the presence or increased level of one or moremicrobes (e.g., one or more types of bacteria) in a subject potentiatescancer/tumor growth, spread (e.g., malignancy), and/or evasion oftreatment/immune response. In some embodiments, the presence orincreased level of one or more microbes (e.g., one or more types ofbacteria) in a subject inhibits treatment (e.g., immunotherapy,chemotherapy, etc.) and/or the subject's endogenous immune response tocancer and/or tumor cells. In some embodiments, the absence and/ordecreased level of one or more microbes (e.g., one or more types ofbacteria) in a subject potentiates cancer/tumor growth, spread, and/orevasion of treatment/immune response. In some embodiments, the absenceor decreased level of one or more microbes (e.g., one or more types ofbacteria) in a subject inhibits treatment (e.g., immunotherapy,chemotherapy, etc.) and/or the subject's endogenous immune response tocancer and/or tumor cells.

In some embodiments, the presence or increased level of one or moremicrobes (e.g., one or more types of bacteria) in a subject discouragescancer/tumor growth, spread, and/or evasion of treatment/immuneresponse. In some embodiments, the presence or increased level of one ormore microbes (e.g., one or more types of bacteria) in a subjectfacilitates treatment (e.g., immunotherapy, chemotherapy, etc.) and/orthe subject's endogenous immune response to cancer and/or tumor cells.In some embodiments, the absence and/or decreased level of one or moremicrobes (e.g., one or more types of bacteria) in a subject discouragescancer/tumor growth, spread, and/or evasion of treatment/immuneresponse. In some embodiments, the absence or decreased level of one ormore microbes (e.g., one or more types of bacteria) in a subjectfacilitates treatment (e.g., immunotherapy, chemotherapy, etc.) and/orthe subject's endogenous immune response to cancer and/or tumor cells.

In some embodiments, the presence of beneficial microbes (e.g., microbesthat facilitate cancer treatment) in a subject creates an environment ormicroenvironment (e.g., metabolome) that is conducive to the treatmentof cancer and/or inhibits cancer/tumor growth. In some embodiments, thepresence of detrimental microbes (e.g., microbes that facilitatecancer/tumor growth and/or prevent treatment) in a subject creates anenvironment or microenvironment (e.g., metabolome) that is conducive tothe treatment of cancer and/or inhibits cancer/tumor growth.

Experiments conducted during development of embodiments described hereindemonstrate that modulation of levels and/or identity of the microflorain a subject facilitates treatment of cancer/tumor within the subject,enhances the endogenous immune response, decreases immune evasion orother inhibitory mechanisms to treatment of endogenous immune response,and/or improves cancer outcomes for the subject. Modulation ofmicroflora levels and/or identity may comprise encouraging orfacilitating growth of one or more types of beneficial microbes (e.g.,microbes that facilitate cancer treatment), discouraging or inhibitinggrowth of one or more types of detrimental microbes (e.g., microbes thatfacilitate cancer/tumor growth and/or prevent treatment), administeringone or more types of beneficial microbes (e.g., microbes that facilitatecancer treatment) to the subject, and/or combinations thereof.

Embodiments within the scope herein are not limited by the mechanismsfor introducing one or more microbes (e.g., fecal transplant, probioticadministration, etc.), encouraging growth of beneficial microbes (e.g.,administering agents that skew the environment within the subject towardgrowth conditions for the beneficial microbes), discouraging orinhibiting growth of detrimental microbes (e.g., administering agentsthat skew the environment within the subject away from growth conditionsfor the detrimental microbes, administration of antimicrobial(s), etc.),and combinations thereof.

In some embodiments, methods are provided for the treatment orprevention of cancer by the manipulation of the presence, amount, orrelative ratio of commensal microflora (e.g., gut microflora). In someembodiments, the presence, amount, or relative ratio of particularbacteria, fungi, and/or archaea within a subject is manipulated. Forexample, in some embodiments, the presence, amount, or relative ratio ofone or more bacteria from the phyla Firmicutes, Bacteroidetes,Actinobacteria, and/or Proteobacteria are manipulated. In someembodiments, the presence, amount, or relative ratio of one or morebacteria belonging to the genera Bacteroides, Clostridium,Fusobacterium, Eubacterium, Ruminococcus, Peptococcus,Peptostreptococcus, Bifidobacterium, Rikenella, Alistipes,Marinilabilia, Anaerostipes, Escherichia, and/or Lactobacillus aremanipulated. In some embodiments, the presence, amount, or relativeratio of one or more fungi belonging to the genus Candida,Saccharomyces, Aspergillus, and/or Penicillium are manipulated.

In some embodiments, the presence and/or levels of one or more commensalmicrobes are manipulated in a subject suffering from cancer, atheightened risk of cancer, and/or receiving treatment for cancer.Exemplary commensal microbes include Lactococcus (e.g., Lactococcuscremoris and Lactococcus lactis), Lactobacillus (e.g., Lactobacillusacidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillusbifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillusparacasei, Lactobacillus rhamnosus, Lactobacillus salivarius,Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei,Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillusfarciminis, Lactobacillus lactis, Lactobacillus delbrueckii,Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacilluscrispatus, Lactobacillus gasseri, Lactobacillus johnsonii andLactobacillus jensenii), Leuconostoc, Carnobacterium, Enterococcus,Propionibacteium, Pediococcus, Bifidobacterium (e.g., Bifidobacteriumlactis, Bifidobacterium bifidium, Bifidobacterium longum,Bifidobacterium animalis, Bifidobacterium breve, Bifidobacteriuminfantis, Bifidobacterium catenulatum, Bifidobacteriumpseudocatenulatum, Bifidobacterium adolescentis,Bifidobacteriumangulatum, etc.), Streptococcus (e.g., Streptococcusthermophiles, Streptococcus salivarius, Streptococcus oralis,Streptococcus uberis, Streptococcus rattus, etc.); Escherichia coli,Bacillus coagulans, Bacillus lansii, Yeast (e.g., Saccharomycescerevisiae, Saccharomyces boulardii, etc.); and combinations thereof.

In some embodiments, one or more species, genera, and/or types ofmicrobes are administered and/or the growth thereof is facilitated. Insome embodiments, the growth of one or more species, genera, and/ortypes of microbes is inhibited. In some embodiments, one or morespecies, genera, and/or types of microbes are administered and/or thegrowth thereof is facilitated; and the growth of one or more otherspecies, genera, and/or types of microbes is inhibited.

In some embodiments, the level or presence of one or more beneficialmicrobes (e.g., microbes that inhibit cancer/tumor growth or spread,enhance cancer/tumor treatment, etc.) is modulated by the administrationof such microbes to a subject.

In some embodiments, microflora-modulation utilizes prepared probioticcompositions for administration to/by a subject. Probiotic compositionscomprise one or more beneficial microbes (e.g., bacteria) formulatedsuch that administration of the probiotic (e.g., orally, rectally, byinhalation, etc.) results in population of the subject by the beneficialmicrobes. In some embodiments, probiotic compositions comprise culturedmicrobes that are combined and/or formulated for administration to asubject. In some embodiments, probiotics contain microbes of knowngenera, species, etc. and/or at known concentrations (cfus). Probioticcompositions may be in the form of a pharmaceutical-type composition(e.g., capsule, tables, liquid, aerosol, etc.) or in the form of a foodsupplement.

In some embodiments, probiotic microbes (e.g., bacteria) are formulatedin a pharmaceutically acceptable composition for delivery to a subject.In some embodiments, probiotics are formulated with a pharmaceuticallyacceptable carrier suitable for a solid or semi-solid formulation. Insome embodiments, probiotic microbes are formulated with apharmaceutically acceptable carrier suitable for a liquid or gelformulation. Probiotic formulations may be formulated for enteraldelivery, e.g., oral delivery, or delivery as a suppository, but canalso be formulated for parenteral delivery, e.g., vaginal delivery,inhalational delivery (e.g., oral delivery, nasal delivery, andintrapulmonary delivery), and the like.

The probiotic compositions that find use in embodiments described hereinmay be formulated in a wide variety of oral administration dosage forms,with one or more pharmaceutically acceptable carriers. Thepharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier can beone or more substances which may also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, preservatives,tablet disintegrating agents, or an encapsulating material. In powders,the carrier is a finely divided solid which is a mixture with theprobiotic microbes. In tablets, the microbes are mixed with the carrierhaving the necessary binding capacity in suitable proportions andcompacted in the shape and size desired. Suitable carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Other forms suitable for oral administration include liquid formpreparations such as emulsions, syrups, elixirs, aqueous solutions,aqueous suspensions, or solid form preparations which are intended to beconverted shortly before use to liquid form preparations. Aqueoussuspensions can be prepared by dispersing the probiotic microbes inwater with viscous material, such as natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, and other well-knownsuspending agents.

The probiotic compositions (e.g., microbes (e.g., bacteria)) may beformulated for administration as suppositories. A low melting wax, suchas a mixture of fatty acid glycerides or cocoa butter is first meltedand the probiotic microbes are dispersed homogeneously, for example, bystirring. The molten homogeneous mixture is then poured intoconveniently sized molds, allowed to cool, and to solidify.

The probiotic compositions (e.g., microbes (e.g., bacteria)) may beformulated for vaginal administration. Pessaries, tampons, creams, gels,pastes, foams or sprays, may contain agents in addition to the bacteria,such carriers, known in the art to be appropriate.

In some embodiments, probiotic compositions (e.g., microbes (e.g.,bacteria)) may be formulated for delivery by inhalation. As used herein,the term “aerosol” is used in its conventional sense as referring tovery fine liquid or solid particles carries by a propellant gas underpressure to a site of therapeutic application. The term “liquidformulation for delivery to respiratory tissue” and the like, as usedherein, describe compositions comprising probiotic microbes with apharmaceutically acceptable carrier in flowable liquid form. Suchformulations, when used for delivery to a respiratory tissue, aregenerally solutions, e.g. aqueous solutions, ethanolic solutions,aqueous/ethanolic solutions, saline solutions and colloidal suspensions.

Rather than pharmaceutical-type formulation, probiotic compositions maybe formulated as food additive and/or food product and incorporated intoa variety of foods and beverages. Suitable foods and beverages include,but are not limited to, yogurts, ice creams, cheeses, baked productssuch as bread, biscuits and cakes, dairy and dairy substitute foods,soy-based food products, grain-based food products, starch-based foodproducts, confectionery products, edible oil compositions, spreads,breakfast cereals, infant formulas, juices, power drinks, and the like.

In some embodiments, a probiotic composition is administered over adosing time period (e.g., <1 minute, <1 hour, <2 hours, <4 hours, <6hours, <12 hours, <24 hours, etc.) in an amount that is sufficient toprovide a desired therapeutic benefit (e.g., as a single dose, incombination with other doses, in combination with a co-administeredtherapeutic, etc.) In some embodiments, the dose of the probioticcomposition administered for the dosing time period is concentration offrom about 10 to about 1×10¹⁴ colony forming units (cfu) of thecommensal microbial agent(s) (e.g., 10 cfu, 100 cfu, 10¹³ cfu, 10¹³ cfu,10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu,10¹³ cfu, 10¹³ cfu, 10¹³ cfu, or any suitable ranges therein (e.g., fromabout 10² cfu to about 10¹³ cfu, about 1×10⁴ to about 1×10¹¹ cfu, about1×10⁶ to about 1×10⁹ cfu, about 1×10¹⁰ to about 1×10¹² cf, etc.), etc.).

In some embodiments, the microbial make-up of a probiotic compositionconsists or consists essentially of one or more beneficial microbes(e.g., bacteria). In some embodiments, the microbial make-up of aprobiotic composition consists or consists essentially of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or anyranges therein (e.g., 1-4, 5-10, 8-20, etc.) strains and/or species ofmicrobes. In some embodiments, fewer than 50 microbial strains (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or any rangestherein (e.g., 1-4, 5-10, 8-20, etc.) are at least 50% (e.g., 50%, 60%,70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%.99.9%, 99.99%) of the microbial population (e.g., by mass, by cfu, etc.)of a probiotic composition. For example, in some embodiments, a singlespecies or strain of Bifidobacterium is at least 95% of the microbialpopulation, as measured by colony forming units, of a particularprobiotic composition. As another example, in some embodiments, a singlespecies or strain of Bifidobacterium is at least 40% and bacteria fromthe genus Lactobacillus are at least 50% of the microbial population, asmeasured by mass, of a particular probiotic composition. These examplesare not limiting.

In some embodiments, microflora in a subject (e.g., a subject sufferingfrom cancer, a subject with microflora that promotes cancer growth, asubject with microflora that promotes evasion of cancer treatment (e.g.,by immunotherapy), etc.) are modulated by transplantation of microbiotafrom a subject with favorable characteristics (e.g., a subject withoutcancer, a subject with microflora that inhibits cancer growth, a subjectwith microflora that promotes treatment of cancer (e.g., byimmunotherapy), etc.) into the recipient subject.

In some embodiments, donor microflora are obtained sampling microflorafrom the desired region of the donor subject body (e.g., colon, oralcavity, vagina, etc.). In particular embodiments, fecal material (e.g.,100 g-500 g) is obtained from a donor. The material may be administeredto a recipient subject with or without subsequent preparation steps(e.g., diluting, mixing, oxygenating, filtering, supplementing (e.g.,with prebiotics, with growth media, etc.), testing (e.g., for pathogensor detrimental microbes), etc.). The donor microflora (e.g., fecalmaterial) may be administered without preservation (e.g., administeredwithin 12 hours (e.g., <6 hours, <4 hours, <2 hours, <1 hour, etc.)) ormay be preserved (e.g., frozen, freeze dried, etc.), for example, toallow for delay (e.g., 1 day, 2, days, 1 week, 1 month, or more) beforedelivery to the subject.

In some embodiments, donor microflora are proceed to remove one or morecomponents. For example, parasitic of detrimental microbes may beremoved or killed. Contaminants within the donor sample may be removed.In some embodiments, donor microflora is enriched for one or morespecific microbes (e.g., 2-fold, 3-fold, 4 fold, 10-fold, 20-fold, ormore enrichment). In some embodiments, donor microflora is enriched suchthat at least 1% of the microbes in the population are the desiredbeneficial microbes (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more). In someembodiments, donor microflora are doped with one or more culturedbeneficial microbes.

In particular embodiments, transplanted microflora may be administeredto the recipient subject by any suitable delivery mechanism, includingbut not limited to enema, colonoscope, nasogastric or nasoduodenal tube,lavage or irrigation, or orally (e.g., in the form of a capsule).

In some embodiments, a commensal microbial agent or population ofmicrobial agents is administered (e.g., via probiotic composition ormicroflora transplant) over a dosing time period (e.g., <1 minute, <1hour, <2 hours, <4 hours, <6 hours, <12 hours, <24 hours, etc.) in anamount that is sufficient to provide a desired therapeutic benefit(e.g., as a single dose, in combination with other doses, in combinationwith a co-administered therapeutic, etc.) In some embodiments, the doseof commensal microbial agent(s) administered for the dosing time periodis concentration of from about 10 to about 1×10¹⁴ colony forming units(cfu) of the commensal microbial agent(s) (e.g., 10 cfu, 100 cfu, 10¹³cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10 ¹³ cfu, 10¹³cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, 10¹³ cfu, or any suitable rangestherein (e.g., from about 10² cfu to about 10¹³ cfu, about 1×10⁴ toabout 1×10¹¹ cfu, about 1×10⁶ to about 1×10⁹ cfu, about 1×10¹⁰ to about1×10¹² cf, etc.), etc.).

The dose can be administered in a single unit dose administered at anytime during a day. Alternatively the loading dose can be administered intwo or more doses administered at a single time of day or at two or moreseparate times of day.

Over the course of multiple dosing periods, the dose can be tapered froman initial dose to a higher dose (or increased from an initial dose to ahigher dose), on predetermined timing or by the when the subject and/orclinician based on the results of the treatment. The appropriate dosageamount will vary by, for example, an individual subject's age, weight,condition or disease, severity of disease, etc.

By way of non-limiting example (both in terms of identify of the microbeas well as dose), in some embodiments, one or more strains ofBifidobacterium are administered via 3 capsules daily, each capsulecontaining 1×10⁹ cfu of Bifidobacterium. Alternatively, in otherembodiments one or more strains of Bifidobacterium are administered at adosage of one capsule daily containing 1×10¹² cfu of bacteria. Any otherdosages (e.g., cfu), doses (e.g., times per day, week, etc.), andidentity of the microbe(s) (e.g., within the ranges described herein)are within the scope herein.

In some embodiments, microbes for probiotic compositions are obtainedfrom culture. In some embodiments, strains of beneficial microbes aregenetically engineered to enhance one or more of production (e.g., atscale), formulation, delivery, or the biological effect of the microbe.In some embodiments, microbes are engineered to express a detectablemarker that allows tracking of the microbes within a subject, orconfirmation that the microbe has integrated into a subjects microflora.In some embodiments, microbes are engineered to express a cancertherapeutic (e.g., chemotherapeutic, immunotherapeutic, antibodies,etc.), anti-inflammatory agent, of other drug.

In some embodiments, one or more prebiotics are administered to asubject as an independent treatment (e.g., to increase the level of abeneficial microbe) or in conjunction with other treatments describedherein. Prebiotics are agents that increase the in vivo growth rate oractivity of commensal microbes, such as Lactobacillus and/orBifidobacterium. In some embodiments, prebiotics are soluble fibersources. In some embodiments, when prebiotics are administered (e.g.,fed) to a subject they are not digested or are not fully digested by thesubject's digestive enzymes, but rather support the intestinal health ofthe subject and provide an energy source for the beneficial microbes andenhance the growth thereof. Prebiotics include, for example, naturallyoccurring lecithins and/or oleic acid, and are described, for example inU.S. Pat. No. 8,449,878 which is herein incorporated by reference in itsentirety.

In some embodiments, the level or presence of one or more detrimentalmicrobes (e.g., microbes that facilitate cancer/tumor growth or spread,inhibit cancer/tumor treatment, etc.) is modulated, for example, by theadministration of one or more antimicrobial agents to a subject ormodulation of conditions within the subject to disfavor growth of thedetrimental microbes. In some embodiments, antimicrobial agents areadministered.

In some embodiments, the antimicrobial agent is an antibiotic. Exemplaryantibiotics that may find use in some embodiments include, but are notlimited to: amikacin, gentamicin, kanamycin, neomycin, netilmicin,streptomycin, tobramycin, paromycin, geldanamycin, herbimycin,loracarbef, ertapenem, doripenem, imipenem, meropenem, cefaclor,cefamandole, cefotoxin, cefprozil, cefuroxime, cefixime, cefdinir,cefditoren, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefepime, ceftobirprole, vancomycin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,troleandomycin, telithromycin, spectinomycin, aztreonam, amoxicillin,ampicillin, azociling, carbenicillin, cloxacillin, dicloxacillin,flucloxacin, mexlocillin, meticillin, nafcillin, oxacillin,peperacillin, ticarcillin, bacitracin, colistin, polymyxin B,ciprofloxacin, clavulanic acid, enoxacin, gatifloxacin, levofloxacin,lomefloxacin, moxifloxacin, nonfloxacin ofloxacin, trovafloxacin,grepafloxacin, sparfloxacin, AL-15469A, AL-38905, OP-145, afenide,prontosil, sulfacetamide, sulfamethiazole, sulfanamide, sulfasalazine,sulfisoxazole, trimethoprim, cotrimoxazole, demeclocycline, doxycycline,minocycline, oxytetracycline, tetraycline, linezolid, arsogebanubemchloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin,fusidic acid, furazolidone, isoniazid, linezolid, metronidazole,mupirocin, nitrofurantoin, rifampicin, thamphenicol, tinidazole,amoxicillin+clavulanic acid, Maximin H5, Dermcidin, Cecropins, andropin,moricin, ceratotoxin, melittin Magainin, dermaseptin, bombinin,brevinin-1, esculentins and buforin II, CAP 18, LL37, abaecin,apidaecins, prophenin, indolicidin, brevinins, protegrin, tachyplesins,defensins, drosomycin, alamethicin, pexiganan or MSI-78, MSI-843,DISI-594, polyphemusin, colicin, pyocin, klebicin, subtilin, epidermin,herbicolacin, brevicin, halocin, agrocin, alveicin, carnocin,curvaticin, divercin, enterocin, enterolysin, erwiniocin, glycinecin,lactococin, lacticin, leucoccin, mesentericin, pediocin, plantaricin,sakacin, sulfolobicin, vibriocin, warnerinand, nisin, or a salt orcocrystal, or prodrug or solvate thereof, or a combination thereof.

In some embodiments, the antimicrobial is an antifungal agent. Exemplaryantifungals that may find use in some embodiments include, but are notlimited to: amrolfine, utenafine, naftifine, terbinafine, flucytosine,fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole,voriconazole, clotrimazole, econazole, miconazole, oxiconazole,sulconazole, terconazole, tioconazole, nikkomycin Z, caspofungin,micafungin, anidulafungin, amphotericin B, liposomal nystastin,pimaricin, griseofulvin, ciclopirox olamine, haloprogin, tolnaftate,undecylenate, clioquinol, and combinations thereof.

In some embodiments, the antimicrobial is an antiparasitic. Exemplaryantiparasitics that may find use in some embodiments include, but arenot limited to: amitraz, amoscanate, avermectin, carbadox,diethylcarbamizine, dimetridazole, diminazene, ivermectin,macrofilaricide, malathion, mitaban, oxamniquine, permethrin,praziquantel, prantel pamoate, selamectin, sodium stibogluconate,thiabendazole, and combinations thereof.

In some embodiments, methods and compositions for reduction ofdetrimental microbe levels are co-administered (e.g., serially,concurrently, etc.) with methods and compositions for increasingbeneficial microbe levels. In some embodiments, by reducing overallmicrobe levels or by reducing the levels of specific microbes (e.g.,detrimental microbes, high population microbes, etc.), the population ofbeneficial microbes can more effectively be modulated (e.g., increased).

In some embodiments, in order to develop a microflora population withina subject that facilitates cancer treatment or inhibits cancergrowth/spread, antimicrobial agents are first administered to eliminateor reduce the microflora within the subject, and then the microflorapopulation is reestablished using the methods and compositions describedherein (e.g., administration of beneficial microbes). In someembodiments, antimicrobials (e.g., antibiotics) that reduce the microbe(e.g., bacteria) population generally are employed. In some embodiments,antimicrobials that target detrimental microbes preferentially areemployed.

In some embodiments, modulating the microflora composition is sufficienton its own to allow the endogenous immune system of a subject to respondto the presence of cancer cells and or tumor growth. However, in otherembodiments, microflora composition is manipulated along with one ormore other cancer therapies. In some embodiments, manipulation of themicroflora composition (e.g., identity and/or level) treats cancer by amechanism independent of one or more additional cancer treatments. Inother embodiments, modulation of microflora composition facilitates(e.g., increases the effectiveness of) the cancer treatment. In someembodiments, one or more cancer treatments enhance the effectiveness ofthe modulation of microflora composition. Embodiments herein are notlimited by the types of cancer treatments (e.g., surgery, radiation,immunotherapy, chemotherapeutic, etc.) unless specifically noted.

In some embodiments, immunotherapeutic cancer treatment encompassesblockade of immune-inhibitory receptors, for example using monoclonalantibodies (mAbs) against CTLA-4 and PD-1/PD-L1 (Wolchok, J. D. et al.The New England Journal of Medicine 369, 122-133 (2013).; Topalian, S.L. et al. Journal of clinical oncology 32, 1020-1030 (2014).; Topalian,S. L. et al. The New England journal of medicine 366, 2443-2454 (2012).;Hodi, F. S. et al. The New England journal of medicine 363, 711-723(2010).; herein incorporated by reference in their entireties).

In some embodiments, the immunotherapy includes the administration of animmune checkpoint inhibitor. Immune Checkpoint inhibition broadly refersto inhibiting the checkpoints that cancer cells can produce to preventor downregulate an immune response. Examples of immune checkpointproteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2,A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. Immune checkpointinhibitors can be antibodies or antigen binding fragments thereof thatbind to and inhibit an immune checkpoint protein. Examples of immunecheckpoint inhibitors include, but are not limited to, nivolumab,pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042,RG-7446, BMS-936559, BMS-936558, MK-3475, CT 011, MPDL3280A, MEDI-4736,MSB-0020718C, AUR-012 and STI-A1010. In some embodiments, the immunecheckpoint inhibitor may be administered via injection (e.g.,intravenously, intratumorally, subcutaneously, or into lymph nodes), butmay also be administered orally, topically, or via aerosol.

In some embodiments, the compositions for and/or methods of modulatingmicroflora in a subject overcome immune invasion of cancer cells, tumor,tumor microenvironment, etc. In some embodiments, one or more additionalcancer immunotherapies are employed (e.g., concurrently or serially) tomake use of the induced immune-responsiveness treated cells/tumor.

Suitable immunotherapies may include, but are not limited to: cell-basedtherapies (e.g., dendritic cell or T cell therapy, etc.), monoclonalantibody (mAb) therapy (e.g., naked mAbs, conjugated mAbs), cytokinetherapy (e.g., interferons, interleukins, etc.), adjuvant treatment(e.g., polysaccharide-K), etc.

Examples of antibodies that may find use in the compositions and methodsdisclosed herein, particularly for use in immunotherapies (but not solimited) include, but are not limited, to antibodies such as trastuzumab(anti-HER2/neu antibody); Pertuzumab (anti-HER2 mAb); cetuximab(chimeric monoclonal antibody to epidermal growth factor receptor EGFR);panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody);Zalutumumab (anti-EGFR mAb); Necitumumab (anti-EGFR mAb); MDX-210(humanized anti-HER-2 bispecific antibody); MDX-210 (humanizedanti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptorbispecific antibody); Rituximab (chimeric murine/human anti-CD20 mAb);Obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb);Tositumumab-1131 (anti-CD20 mAb); Ibritumomab tiuxetan (anti-CD20 mAb);Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); Ranibizumab(anti-VEGF mAb); Aflibercept (extracellular domains of VEGFR1 and VEGFR2fused to IgG1 Fc); AMG386 (angiopoietin-1 and -2 binding peptide fusedto IgG1 Fc); Dalotuzumab (anti-IGF-1R mAb); Gemtuzumab ozogamicin(anti-CD33 mAb); Alemtuzumab (anti-Campath-1/CD52 mAb); Brentuximabvedotin (anti-CD30 mAb): Catumaxomab (bispecific mAb that targetsepithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb);Girentuximab (anti-Carbonic anhydrase ix); or Farletuzumab (anti-folatereceptor). Other examples include antibodies such as Panorex™ (17-1A)(murine monoclonal antibody); Panorex (@(17-1A)) (chimeric murinemonoclonal antibody); BEC2 (ami-idiotypic mAb, mimics the GD epitope)(with BCG); Oncolym (Lym-1 monoclonal antibody); SMART M195 Ab,humanized 13′ 1 LYM-1 (Oncolym). Ovarex (B43.13, anti-idiotypic mousemAb); 3622W94 mAb that binds to EGP40 (17-1A) pancarcinoma antigen onadenocarcinomas; Zenapax (SMART Anti-Tac (IL-2 receptor); SMART M195 Ab,humanized Ab, humanized); NovoMAb-G2 (pancarcinoma specific Ab); TNT(chimeric mAb to histone antigens); TNT (chimeric mAb to histoneantigens); Gliomab-H (Monoclonals-Humanized Abs); GNI-250 Mab; EMD-72000(chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); andMDX-260 bispecific, targets GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364Ab, or ImmuRAIT-CEA.

In some embodiments, an immunotherapy, utilized as a co-therapy with themicroflora modulation described herein, directly or indirectly targetsone of more of: a regulatory T cell, myeloid suppressor cell, ordendritic cell. In another aspect, an immunotherapy specifically targetsone of the following molecules: CD4; CD25 (IL-2α receptor; IL-2αR);cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152); Interleukin-10(IL-10); Transforming growth factor-beta receptor (TGF-βR); Transforminggrowth factor-beta (TGF-β); Programmed Death-1 (PD-1); Programmeddeath-1 ligand (PD-L1 or PD-L2); Receptor activator of nuclear factor-κB(RANK); Receptor activator of nuclear factor-κB (RANK) ligand (RANKL);LAG-3; glucocorticoid-induced tumor necrosis factor receptorfamily-related gene (GITR; TNFRSF18); or Interleukin-4 receptor (IL-4R).In some embodiments, the immunotherapy acts as an agonist that increasesthe function of the targeted molecule. In other embodiments, theimmunotherapy is an antagonist that inhibits the function of thetargeted molecule.

In some embodiments, an immunotherapy, utilized as a co-therapy with themicroflora modulation described herein, directly or indirectly targetsone of more of a specific cytokine, cytokine receptor, co-stimulatorymolecule, co-inhibitory molecule, or immunomodulatory receptor thatmodulates the immune system. In another aspect, one of the followingmolecules are targeted by co-treatment with microflora modulation: tumornecrosis factor (TNF) superfamily; tumor necrosis factor-α (TNF-α);tumor necrosis factor receptor (TNFR) superfamily; Interleukin-12(IL-12); IL-12 receptor; 4-1BB (CD137); 4-1BB ligand (4-1BBL; CD137L);OX40 (CD134; TNR4); OX40 ligand (OX4OL; CD40; CD40 ligand (CD40L);CTLA-4; Programmed death-1 (PD-1); PD-1 ligand I (PD-L1: B7-H1); or PD-1ligand 2 (PD-L2; B7-DC); B7 family; B7-1 (CD80); B7-2 (CD86); B7-H3;B7-H4; GITR/AITR: GITRL/AITRL; BTLA; CD70; CD27; LIGHT; HVEM: Toll-likereceptor (TLR) (TLR 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In some embodiments, the compositions for and/or methods of modulatingmicroflora in a subject sensitize the cancer cells and/or tumor totreatment by one or more chemotherapeutic agents. In some embodiments,one or more chemotherapies are employed in addition to microfloramodulation (e.g., concurrently or serially) to make use of the inducedchemotherapeutic sensitivity. In other embodiments, one or morechemotherapeutics are provided as co-therapies with microfloramodulation, with or without (known) synergism between the microfloramodulation and the chemotherapy.

In some embodiments, exemplary anticancer agents suitable for use incompositions and methods described herein (e.g., co-administered with aβ-catenin inhibitor) include, but are not limited to: 1) alkaloids,including microtubule inhibitors (e.g., vincristine, vinblastine, andvindesine, etc.), microtubule stabilizers (e.g., paclitaxel (Taxol), anddocetaxel, etc.), and chromatin function inhibitors, includingtopoisomerase inhibitors, such as epipodophyllotoxins (e.g., etoposide(VP-16), and teniposide (VM-26), etc.), and agents that targettopoisomerase I (e.g., camptothecin and isirinotecan (CPT-11), etc.); 2)covalent DNA-binding agents (alkylating agents), including nitrogenmustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide,ifosphamide, and busulfan (MYLERAN), etc.), nitrosoureas (e.g.,carmustine, lomustine, and semustine, etc.), and other alkylating agents(e.g., dacarbazine, hydroxymethylmelamine, thiotepa, and mitomycin,etc.); 3) noncovalent DNA-binding agents (antitumor antibiotics),including nucleic acid inhibitors (e.g., dactinomycin (actinomycin D),etc.), anthracyclines (e.g., daunorubicin (daunomycin, and cerubidine),doxorubicin (adriamycin), and idarubicin (idamycin), etc.),anthracenediones (e.g., anthracycline analogues, such as mitoxantrone,etc.), bleomycins (BLENOXANE), etc., and plicamycin (mithramycin), etc.;4) antimetabolites, including antifolates (e.g., methotrexate, FOLEX,and MEXATE, etc.), purine antimetabolites (e.g., 6-mercaptopurine (6-MP,PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir,chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and2′-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g.,fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine(FdUrd) (floxuridine)) etc.), and cytosine arabinosides (e.g., CYTOSAR(ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase,and hydroxyurea, etc.; 6) hormones, including glucocorticoids,antiestrogens (e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g.,flutamide, etc.), and aromatase inhibitors (e.g., anastrozole(ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatin andcarboplatin, etc.); 8) monoclonal antibodies (e.g., conjugated withanticancer drugs, toxins, and/or radionuclides, etc.; neutralizingantibodies; etc.); 9) biological response modifiers (e.g., interferons(e.g., IFN-.alpha., etc.) and interleukins (e.g., IL-2, etc.), etc.);10) adoptive immunotherapy; 11) hematopoietic growth factors; 12) agentsthat induce tumor cell differentiation (e.g., all-trans-retinoic acid,etc.); 13) gene therapy techniques; 14) antisense therapy techniques;15) tumor vaccines; 16) therapies directed against tumor metastases(e.g., batimastat, etc.); 17) angiogenesis inhibitors; 18) proteosomeinhibitors (e.g., VELCADE); 19) inhibitors of acetylation and/ormethylation (e.g., HDAC inhibitors); 20) modulators of NF kappa B; 21)inhibitors of cell cycle regulation (e.g., CDK inhibitors); and 22)modulators of p53 protein function.

In some embodiments, compositions and methods herein comprise multiplemodes for the treatment and/or prevention of cancer. In someembodiments, beneficial microbes are provided/administered (e.g., by aprobiotic composition, fecal transplant, etc.) with prebiotics and/orother agents that facilitate the growth of the beneficial microbes. Insome embodiments, beneficial microbes are provided/administered (e.g.,by a probiotic composition, fecal transplant, etc.) withantimicrobial(s) (e.g., antibiotics) directed to kill or inhibit thegrowth of detrimental microbes. In some embodiments, prebiotics and/orother agents that facilitate the growth of the beneficial microbes areprovided/administered with antimicrobial(s) (e.g., antibiotics) directedto kill or inhibit the growth of detrimental microbes. In someembodiments, beneficial microbes, prebiotics and/or other agents thatfacilitate the growth of the beneficial microbes, and anantimicrobial(s) (e.g., antibiotics) directed to kill or inhibit thegrowth of detrimental microbes are all co-administered.

In some embodiments, the co-administered agents are formulated into asingle dose and/or composition. In some embodiments, the co-administeredagents are in separate doses and/or compositions. In some embodiments inwhich separate doses and/or compositions are administered, the dosesand/or compositions are administered simultaneously, consecutively, orspaced over a time span (e.g., <30 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week,or more, or any suitable ranges therebetween).

In some embodiments, beneficial microbes, prebiotics and/or other agentsthat facilitate the growth of the beneficial microbes, antimicrobial(s)(e.g., antibiotics) directed to kill or inhibit the growth ofdetrimental microbes, or any of the above mentioned combinations thereofare administered with a treatment for cancer. In embodiments, in whichthe modulation of microflora itself provides treatment for cancer,suitable co-treatments include immunotherapy, chemotherapy, surgery(e.g., tumor removal), radiation, etc. In other embodiments, in whichthe modulation of microflora sensitizes a subject or the tumormicroenvironment to a particular cancer therapy (e.g., an immunotherapy,a chemotherapy, etc.), the particular cancer therapy is administered(e.g., optionally in addition to one or more other cancer therapies towhich the subject is not directly sensitized to by the modulation).

In some embodiments, microflora modulation is provided as a co-therapy(e.g., chemotherapy, immunotherapy, etc.) with one or more additionaltherapies that target and/or bind to specific cancer or tumor cellmarkers. Such markers may be selected from the group including but notlimited to, epidermal growth factor receptor (EGFR, EGFR1, ErbB-1,HER1). ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family;insulin-like growth factor receptor (IGFR) family, IGF-binding proteins(IGFBPs), IGFR ligand family (IGF-1R); platelet derived growth factorreceptor (PDGFR) family, PDGFR ligand family; fibroblast growth factorreceptor (FGFR) family, FGFR ligand family, vascular endothelial growthfactor receptor (VEGFR) family, VEGF family; HGF receptor family: TRKreceptor family; ephrin (EPH) receptor family: AXL receptor family;leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family,angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR)receptor family; discoidin domain receptor (DDR) family; RET receptorfamily; KLG receptor family; RYK receptor family; MuSK receptor family;Transforming growth factor alpha (TGF-α), TGF-α receptor; Transforminggrowth factor-beta (TGF-β), TGF-β receptor; Interleukin f3 receptoralpha2 chain (IL13Ralpha2), Interleukin-6 (IL-6), 1L-6 receptor,interleukin-4, IL-4 receptor, Cytokine receptors, Class I (hematopoietinfamily) and Class II (interferon/1L-10 family) receptors, tumor necrosisfactor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptorsuperfamily (TNTRSF), death receptor family, TRAIL-receptor;cancer-testis (CT) antigens, lineage-specific antigens, differentiationantigens, alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson(Bcr-abl) fusion products, B-RAF, caspase-5 (CASP-5), caspase-8(CASP-8), beta-catenin (CTNNB1), cell division cycle 27 (CDC27),cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein,EFTUD-2, Elongation factor 2 (ELF2), Ets variant gene 6/acute myeloidleukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB,low density lipid receptor/GDP-L fucose: beta-Dgalactose2-alpha-Lfucosyltraosferase (LDLR/FUT) fusion protein, HLA-A2, MLA-A11,heat shock protein 70-2 mutated (HSP70-2M), KIAA0205, MART2, melanomaubiquitous mutated 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase(PAP), neo-PAP, Myosin class 1, NFYC, OGT, OS-9, pml-RARalpha fusionprotein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600,SIRT12, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TriosephosphateIsomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8,GnT-V (aberrant N-acetyl glucosaminyl transferase V, MGAT5), HERV-K MEL,KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma(CAMEL), MAGE-A1 (MAGE-1). MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6,MAGE-A8, MAGE-A9, MAGE-A10. MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1,MAGE-B2, MAGE-B5. MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1),MART-1/Melan-A (MLANA), gp100, gp100/Pme117 (S1LV), tyrosinase (TYR),TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE, Sp17. SSX-1, 2, 3,4, TRP2-1NT2, carcino-embryonic antigen (CEA), Kallikrein 4,mammaglobin-A, OA1, prostate specific antigen (PSA), prostate specificmembrane antigen, TRP-1/, 75. TRP-2 adipophilin, interferon inducibleprotein absent in melanoma 2 (AIM-2). BING-4, CPSF, cyclin D1,epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growthfactor-5 (FGF-5), glycoprotein 250 (gp250intestinal carboxyl esterase(iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUCI, p53 (TP53), PBF,PRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, survivin (BIRCS),human telomerase reverse transcriptase (hTERT), telomerase, Wilms' tumorgene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1,CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC,SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE,immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors (ER),androgen receptors (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3,cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancerantigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen19-9 (CA 19-9), beta-human chorionic gonadotropin, 1-2 microglobulin,squamous cell carcinoma antigen, neuron-specific enolase, heat shockprotein gp96. GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP),adenocarcinoma antigen recognized by T cells 4 (ART-4),carcinoembryogenic antigen peptide-1 (CAP-1), calcium-activated chloridechannel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2(HST-2), etc.

Non-limiting examples of cancers that may be treated with thecompositions and methods described herein include, but are not limitedto: cancer cells from the bladder, blood, bone, bone marrow, brain,breast, colon, esophagus, gastrointestine, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,or uterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; and roblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia. In some embodiments, the cancer is a melanoma (e.g.,metastatic malignant melanoma), renal cancer (e.g. clear cellcarcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), pancreatic cancer (e.g., adenocarcinoma), breastcancer, colon cancer, gallbladder cancer, lung cancer (e.g. non-smallcell lung cancer), esophageal cancer, squamous cell carcinoma of thehead and neck, liver cancer, ovarian cancer, cervical cancer, thyroidcancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplasticmalignancies. In some embodiments, the cancer is a solid tumor cancer.

In some embodiments, the methods provided herein relate to the treatmentand/or prevention of a leukemia. The term “leukemia” is meant broadlyprogressive, malignant diseases of the hematopoietic organs/systems andis generally characterized by a distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Non-limiting examples of leukemia diseases include, acute nonlymphocyticleukemia, chronic lymphocytic leukemia, acute granulocytic leukemia,chronic granulocytic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, a leukocythemic leukemia,basophilic leukemia, blast cell leukemia, bovine leukemia, chronicmyelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilicleukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia,stem cell leukemia, subleukemic leukemia, undifferentiated cellleukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblasticleukemia, histiocytic leukemia, stem cell leukemia, acute monocyticleukemia, leukopenic leukemia, lymphatic leukemia, lymphoblasticleukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoidleukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblastic leukemia, myelocytic leukemia, myeloid granulocyticleukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cellleukemia, plasmacytic leukemia, and promyelocytic leukemia.

In some embodiments, the methods provided herein relate to the treatmentand/or prevention of a carcinoma. The term “carcinoma” refers to amalignant growth made up of epithelial cells tending to infiltrate thesurrounding tissues, and/or resist physiological and non-physiologicalcell death signals and gives rise to metastases. Non-limiting exemplarytypes of carcinomas include, acinar carcinoma, acinous carcinoma,adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,carcinoma of adrenal cortex, alveolar carcinoma, alveolar cellcarcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiennoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma,solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma,carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma,string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes,transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma,verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypernephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, and carcinomascroti.

In some embodiments, the methods provided herein relate to the treatmentand/or prevention of a sarcoma. The term “sarcoma” generally refers to atumor which is made up of a substance like the embryonic connectivetissue and is generally composed of closely packed cells embedded in afibrillar, heterogeneous, or homogeneous substance. Sarcomas include,but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromalsarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giantcell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolarsoft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromasarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

Additional exemplary neoplasias that can be treated and/or preventedusing the methods described herein include Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer.

In some embodiments, the cancer treated and/or prevented is a melanoma.The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Non-limiting examplesof melanomas are Harding-Passey melanoma, juvenile melanoma, lentigomaligna melanoma, malignant melanoma, acral-lentiginous melanoma,amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91melanoma, nodular melanoma subungal melanoma, and superficial spreadingmelanoma.

Particular categories of tumors that can be treated and/or preventedusing methods described herein include lymphoproliferative disorders,breast cancer, ovarian cancer, prostate cancer, cervical cancer,endometrial cancer, bone cancer, liver cancer, stomach cancer, coloncancer, pancreatic cancer, cancer of the thyroid, head and neck cancer,cancer of the central nervous system, cancer of the peripheral nervoussystem, skin cancer, kidney cancer, as well as metastases of all theabove. Particular types of tumors include hepatocellular carcinoma,hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma,thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma,invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonarysquamous cell carcinoma, basal cell carcinoma, adenocarcinoma (welldifferentiated, moderately differentiated, poorly differentiated orundifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma,hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testiculartumor, lung carcinoma including small cell, non-small and large celllung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma,colon carcinoma, rectal carcinoma, hematopoietic malignancies includingall types of leukemia and lymphoma including: acute myelogenousleukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia,multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma.

Cancers prevented and/or treated in certain embodiments also includeprecancerous lesions, e.g. actinic keratosis (solar keratosis), moles(dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns,Barrett's esophagus, atrophic gastritis, dyskeratosis congenita,sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic(solar) elastosis and cervical dysplasia.

Cancers prevented and/or treated in some embodiments includenon-cancerous or benign tumors, e.g. of endodermal, ectodermal ormesenchymal origin, including, but not limited to cholangioma, colonicpolyp, adenoma, papilloma, cystadenoma, liver cell adenoma, hydatidiformmole, renal tubular adenoma, squamous cell papilloma, gastric polyp,hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma,leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, andganglioneuroma.

Some embodiments described herein are particularly useful for thetreatment of tumors that do not otherwise respond to immunotherapeuticapproaches. In some embodiments, such tumors are non-responsive (or havea reduced response) to T cells (e.g., prevent infiltration of one ormore T cell types (e.g., CD8⁺ T cells) or antigen presenting cells(e.g., dendritic cells (e.g., CD103⁺ DCs, etc.), etc.). In someembodiments, compositions and methods described herein find use in thetreatment of cancers in which T cells are not appropriately primedagainst tumor-associated antigens.

In some embodiments, methods are provided for testing sample (e.g.,cell, tissue, population of cells, tumor, blood, urine, saliva, etc.)from a subject for one or more biomarkers of cancer, immune evasion,cancer promoting microenvironment, malignancy-promotingmicroenvironment, etc. Such biomarkers may comprise nucleic acids, smallmolecules, proteins, peptides, etc., and may be detected using anysuitable assay of technique. In some embodiments, provided herein areDNA-, RNA-, small molecule, and/or protein-based diagnostic methods thateither directly or indirectly detect the biomarkers of the evasion ofimmune response or immunotherapy by cancer cells or tumors. The presentinvention also provides compositions, reagents, and kits for suchdiagnostic purposes.

In some embodiments, biomarkers are detected at the nucleic acid (e.g.,RNA) level. For example, the presence or amount of biomarker nucleicacid (e.g., mRNA) in a sample is determined (e.g., to determine thepresence or level of biomarker expression). Biomarker nucleic acid(e.g., RNA, amplified cDNA, etc.) may be detected/quantified using avariety of nucleic acid techniques known to those of ordinary skill inthe art, including but not limited to nucleic acid sequencing, nucleicacid hybridization, nucleic acid amplification (e.g., by PCR, RT-PCR,qPCR, etc.), micorarray, Southern and Northern blotting, sequencing,etc. Non-amplified or amplified nucleic acids can be detected by anyconventional means. For example, in some embodiments, nucleic acids aredetected by hybridization with a detectably labeled probe andmeasurement of the resulting hybrids. Nucleic acid detection reagentsmay be labeled (e.g., fluorescently) or unlabeled, and may by free insolution or immobilized (e.g., on a bead, well, surface, chip, etc.).

In some embodiments, biomarkers are detected at the protein level. Forexample, the presence or amount of biomarker protein in a sample isdetermined (e.g., to determine the presence or level of biomarkerexpression or localization). In some embodiments, reagents are providedfor the detection and/or quantification of biomarker proteins. Suitablereagents include primary antibodies (e.g., that bind to the biomarkers),secondary antibodies (e.g., that bind primary antibodies), antibodyfragments, aptamers, etc. Protein detection reagents may be labeled(e.g., fluorescently) or unlabeled, and may by free in solution orimmobilized (e.g., on a bead, well, surface, chip, etc.).

In some embodiments, kits are provided comprising, for example, theprobiotic or microflora transplant compositions described herein. Kitsmay further comprise instructions, cancer treatments, other probiotics,agents to enhance integration of microbes into the subject's microflora,etc.

EXPERIMENTAL Example 1

Materials and Methods:

Animals and tumor model: C57BL/6 mice were obtained from Jacksonlaboratory or Taconic farms. 6-8-week-old female mice were used. TheC57BL/6-derived melanoma cell line B16.F10.SIY (referred to herein asB16.SIY) was generated (Blank et al. Cancer research 64, 1140-1145(2004).; herein incorporated by reference in its entirety). For tumorgrowth experiments, mice were injected subcutaneously with 1×10⁶ B16.SIYtumor cells. Tumor size was measured twice a week until endpoint andtumor volume was determined as length×width×0.5. For B16 parental tumormodel experiments, mice were injected subcutaneously with 1×10⁶ B16.F10tumor cells. For bladder cancer model experiments, mice were injectedsubcutaneously with 2×10⁶ MB49 cells. All experimental animal procedureswere approved by the University of Chicago Animal Care and Use Committee(IACUC).

IFN-γ ELISPOT and SIY Pentamer analyses: Elispot plates (Millipore, MAIPS4510) were coated with purified αIFN-γ (BD) overnight at 4° C. Plateswere blocked with 10% FBS in DMEM for 2 hours at room temperature. Wholesplenocytes were plated at 10⁶ cells per well and stimulated with SIYpeptide overnight at 37° C. Spots were developed using the BD mouseIFN-γ kit (Cat. No. 552569), and the number of spots was measured usingan Immunospot Series 3 Analyzer and analyzed using ImmunoSpot software(Cellular Technology). For pentamer staining, cells were labeled withPE-MHC class I pentamer (Proimmune) consisting of murine H-2K^(b)complexed to SIYRYYGL (SIY) peptide or to control SIINFEKL peptide, andstained with CD3-AX700 (Ebioscience, 17A2), CD8-PacBlue (Biolegend,53-6.7), CD4-APC (Pharmingen, RM4-5), CD62L-PECy7 (Ebioscience, MEL-14),CD44-FITC (BD, IM7) and Fixable Viability-ef780 (Ebioscience). Stainedcells were analyzed using an LSR II cytometer with FACSDiva software(BD). Data analysis was conducted with FlowJo software (Tree Star).

Fecal transfers and αPD-L1 mAb immunotherapy: Fecal pellets from JAX andTAC-derived mice were collected upon arrival in our facility and eachfecal pellet was resuspended in 1 ml of phosphate-buffered saline (PBS).The supernatant from each fecal pellet was used for oral gavage of tworecipient mice, 100 μl per gavage. For prophylactic fecal transferexperiments mice were gavaged with JAX or TAC fecal suspensions once aweek for two weeks prior to tumor inoculation. For therapeutic fecaltransfer experiments, mice were gavaged on days 7 and 14 post tumorimplantation. For combination therapy experiments, mice wereadditionally injected intraperitoneally with 100 μg αPD-L1 mAb(BioXCell) in 100 μl PBS on days 7, 10, 13 and 16 post-tumorimplantation.

Microbial DNA analysis: Bacterial DNA was extracted from murine fecalpellets using PowerSoil®-htp 96 Well Soil DNA Isolation Kit (MoBio cat.#12955-4). The V4-V5 region of the 16S rRNA encoding gene was amplified(earthmicrobiome.org/emp-standard-protocols/; Earth Microbiome Project,2011) and sequenced at the High-Throughput Genome Analysis Core atArgonne National Laboratory. Quantitative Insights Into MicrobialEcology (QIIME) was used to trim and classify sequences (Caporaso et al.Bioinformatics 26, 266-267 (2010).; herein incorporated by reference inits entirety); specifically, the open reference OTU picking protocol wasused at 97% sequence identity against the Greengenes database (05/13release)(McDonald et al. The ISME journal 6, 610-618 (2012).; hereinincorporated by reference in its entirety). PYNAST was used to alignsequences (Caporaso et al. Nat Meth 7, 335-336 (2010).; hereinincorporated by reference in its entirety) and RDP Classifier was usedfor taxonomic assignment (Wang et al. Appl Environ Microbiol 73,5261-5267 (2007).; herein incorporated by reference in its entirety).Community structure was compared using weighted and unweighted UniFracdistances (Lozupone et al. Appl Environ Microbiol 71, 8228-8235 (2005).;herein incorporated by reference in its entirety). G-test was performedto determine differences in bacterial taxa occurrence between fecalcommunities. Prinicipal Coordinate Analysis (PCoA) ordination weregenerated to visually compare beta diversity and Analysis of Similarity(ANOSIM) test statistics were performed to statistically compare within-to between-group similarity in QIIME.

Bacterial administration and heat inactivation: A cocktail oflyophilized Bifidobacterium species (B. bifidum, B. longum, B. lactisand B. breve, Seeking Health) were resuspended in PBS at 5×10⁹ CFU/ml.Each mouse was given 200 μl of Bifidobacterium (1×10⁹ CFU/mouse) by oralgavage 7 and 14 days following tumor inoculation. Heat inactivation wasperformed by boiling rehydrated bifidobacteria at 100° C. for 2 hours.Heat-treated and live bifidobacteria were serially diluted in reducedPBS and plated on reduced clostridial medium (RCM) agar in anaerobicconditions. Plates were subsequently incubated in an anaerobic chamberfor three days to test efficacy of killing. Lactobacillus murinus wascultured in MRS broth overnight, then washed and resuspended in PBS at5×10¹⁰ CFU/ml. Each mouse was orally gavaged with 100 μl of bacterialsuspension (5×10⁹ CFU/mouse) 7 and 14 days following tumor inoculation.

CFSE-labeled 2C CD8+ T cell adoptive transfer: CD8⁺ T cells wereisolated from the spleen and lymph node of naïve CD45.1/.2⁺2C TCR Tgmice using the MACS CD8 T cell Isolation Kit (Miltenyi, Cat No.130-095-236), labeled with 2.5 mM CFSE and injected i.v. into CD45.2⁺C57BL/6 mice derived from either JAX or TAC. 24 hours later, mice wereinoculated with 1×10⁶ B16.SIY melanoma cells s.c. Seven dayspost-adoptive T cell transfer, spleen and tumor-draining lymph node wereharvested and restimulated ex-vivo with SIY peptide in the presence ofbrefeldin A. Samples were stained with Fixable Viability-ef780(Ebioscience), CD45.1-PerCPCy5.5 (Ebioscience, E20), CD45.2-APC(Ebioscience, 104), CD3-AX700 (Ebioscience, 17A2), CD8-BV711 (Biolegend,53-6.7), CD4-BV605 (Biolegend, RM4-5) and IFN-γ-PE (BD, XMG1.2).Intracellular IFN-γ production and CFSE dilution were assessed in gatedCD45.1/.2⁺2C CD8⁺ T cells by flow cytometry.

Dendritic cell sorting and gene expression profiling: TAC mice weregavaged with Bifidobacterium once a week for two weeks.Bifidobacterium-fed mice, newly arrived JAX mice, and newly arrived TACmice were inoculated subcutaneously in both flanks with 5×10⁶DRAQ5-labeled B16.SIY tumor cells. 40 hrs following tumor implantation,whole tumors including infiltrating immune cells were digested incollagenase (Worthington) and filtered into single cell suspensions.Samples from 5 mice in each group were pooled and subsequently stainedwith Fixable Viability-ef506 (Ebioscience), CD45-AF488 (Bioloegend,30-F11), CD3-ef450 (Ebioscience, 145-2C11), CD19-PB (Ebioscience, 1D3),I-A/I-E-PECy7 (Biolegend, M5/114.15.2), CD11c-PE (Ebioscience, N418) andCD11b-PerCpCy5.5 (BD, MJ/70). Live CD45⁺CD3⁻CD19⁻MHCII^(hi)CD11c⁺dendritic cells were sorted directly into RLT Buffer (Qiagen) usingFACSAriaIII (BD) and stored immediately on dry ice. Total RNA wasisolated using RNeasy® Micro kit (Qiagen). RNA was submitted to theFunctional Genomics Facility at the University of Chicago for geneexpression profiling. RNA integrity and concentration were assessedusing an Agilent Bioanalyzer 2100, and all RNA samples used formicroarray analysis had an RNA Integrity Number >9.0. Total RNA wasprocessed into biotinylated cRNA using the Epicentre TargetAmp™ 2-RoundBiotin-aRNA Amplification Kit 3.0 (TAB2R71024). The cRNA was hybridizedto Illumina MouseRef8v2 arrays using Illumina provided protocols andscanned using an Illumina HiScan. Quantile normalized and backgroundsubtracted values were subsequently analyzed using R. Genes whoseexpression value was under 10 were removed from the analysis. Meanfold-change in gene transcript levels between JAX samples relative toTAC, and BIF samples relative to TAC were calculated, and genes whosefold change was over 1.5 in both comparisons (761 gene transcripts) wereinputted into The Database for Annotation, Visualization and IntegratedDiscovery (DAVID) v6.7 for pathway analysis. Genes found to besignificantly enriched (p<0.05) for immune function were then plotted ina heatmap using R software.

Statistical analysis: Tumor growth curves were analyzed using two-wayANOVA, with either Sidak's multiple comparisons posttest for comparisonof two groups, or Tukey's multiple comparisons posttest for comparisonof more than two groups. For comparisons other than tumor growth, MannWhitney's non-parametric T-test was used when comparing two groups andone-way ANOVA with Tukey's multiple comparisons posttest was used whencomparing more than two groups. P<0.05 was considered statisticallysignificant and denoted as follows: *p<0.05, **p<0.01, ***p<0.001,****p<0.0001. Statistical analysis was performed using GraphPad PRISM.

Example 2

Results

Experiments were conducted during development of embodiments of thepresent invention to test whether differences in the specificcomposition of the normal microbiota influence the immune response to agrowing tumor in vivo. Subcutaneous B16.SIY melanoma growth was observedin genetically similar C57BL/6 mice derived from two different mousefacilities, Jackson Laboratory (JAX) and Taconic Farms (TAC), which havebeen shown to differ in their commensal microbes (Ivanov et al. Cell139, 485-498 (2009).; herein incorporated by reference in its entirety).It was found that JAX and TAC mice exhibited significant differences inB16.SIY melanoma growth rate, with tumors growing more aggressively inTAC mice (FIG. 1A). To evaluate whether this difference wasimmune-mediated, tumor antigen-specific T cell responses, as well as Tcell accumulation in the tumor microenvironment, were assessed. In fact,tumor-specific T cell responses were significantly higher in JAX mice(FIGS. 1B and 1C), and markedly increased numbers of tumor-infiltratingT cells were observed (FIG. 1D). To begin to address whether thisdifference could be mediated by commensal microbiota, JAX and TAC micewere co-housed for 3 weeks prior to tumor implantation. It was foundthat cohousing ablated the differences in tumor growth (FIG. 1E) andimmune responses (FIG. 1F-H) between the two mouse populations, arguingfor an environmental influence. Notably, TAC mice appeared to acquirethe JAX phenotype upon cohousing, indicating that JAX mice might becolonized by commensal microbes that dominantly facilitate improvedanti-tumor immunity.

To directly test the role of commensal bacteria in regulating anti-tumorimmunity, JAX fecal suspensions or control TAC fecal suspensions weretransferred into TAC recipients by oral gavage prior to tumorimplantation FIG. 5A). Strikingly, it was found that prophylactictransfer of JAX fecal material into TAC recipients was sufficient todelay tumor growth (FIG. 2A) and enhance induction and infiltration oftumor-specific CD8⁺ T cells (FIGS. 2B-C and 5B), supporting a microbe-or microbial product-derived effect. Reciprocal transfer of TAC fecalmaterial into JAX recipients resulted in only a minimal increase intumor growth rate and did not significantly alter anti-tumor T cellresponses (FIGS. 2A-C and FIG. 5B).

To test whether manipulation of the microbial community could beeffective as a therapy, we administered JAX fecal material alone or incombination with antibodies targeting PD-L1 (αPD-L1) to TAC mice bearingestablished tumors. Transfer of JAX fecal material alone resulted insignificantly slower tumor growth (FIG. 2D), accompanied by increasedtumor-specific T cell responses (FIG. 2E) and infiltration ofantigen-specific T cells into the tumor (FIG. 2F), to the same degree astreatment with systemic αPD-L1 mAb. Combination treatment with both JAXfecal transfer and αPDL1 mAb improved tumor control (FIG. 2D) andcirculating tumor antigen-specific T cell responses (FIG. 2E), whilethere was little additive effect on accumulation of activated T cellswithin the tumor microenvironment (FIG. 2F). Consistent with theseresults, αPD-L1 therapy alone was significantly more efficacious in JAXmice compared to TAC mice (FIG. 2G), which paralleled improvedanti-tumor T cell responses (FIG. 5C). These data indicate that thecommensal microbial composition can influence spontaneous anti-tumorimmunity as well as response to immunotherapy with αPD-L1 mAb.

To identify specific bacteria associated with protective anti-tumorimmune responses, the fecal bacterial content in mice obtained from TACmice, JAX mice, and JAX-fed and TAC-fed TAC mice we compared using the16S ribosomal RNA (rRNA) miSeq Illumina platform. Overall, 933.9±55.2taxa were identified in TAC mice and 653.4±60 taxa were identified inJAX mice, demonstrating decreased species diversity in mice obtainedfrom JAX. TAC mice that were orally administered JAX fecal materialshowed decreased taxa diversity (706.6±117.9, p=0.006) similar to JAXmice, whereas TAC mice that were administered TAC fecal material did notshow altered diversity (895.7±118, p=1, FIG. 3A). Principal coordinateanalysis revealed that fecal samples analyzed from TAC mice thatreceived JAX fecal material co-clustered separately from samples fromcontrol TAC mice and were more similar to samples obtained from JAX mice(FIG. 3B, and became similar to samples obtained from sham and JAXfeces-inoculated JAX mice (FIG. 8A). In contrast, TAC-inoculated TACmice did not change in community diversity relative to sham-inoculatedTAC mice (p=0.4, ANOSIM). Analysis of similarity confirmed that TAC micefed with JAX fecal material were more similar to each other than to TACmice that were given TAC fecal material (p=0.008) or mice obtained fromTAC (p=0.002). Reciprocal transfer of TAC fecal material into JAX hostsresulted in a statistically significant change in community diversity(p=0.003, ANOSIM), yet the distance of the microbial shift was smaller(FIG. 8A).

Comparative analysis of specific bacterial taxa showed that 97 taxa weresignificantly more abundant in JAX mice relative to TAC mice (FDR<0.05)(FIG. 8B), and 51 taxa were significantly increased in JAX-fed TAC micerelative to TAC-fed TAC mice (p<0.05). Only 32 taxa overlapped betweenthese two comparisons, such that they were of greater abundance in bothJAX mice and JAX-fed TAC mice. A significant association was observedfor Bifidobacterium, which showed a positive association with anti-tumorT cell responses and increased in relative abundance over 400-fold inJAX-fed TAC mice (FIG. 8C). Members belonging to several of these groupswere similarly altered in JAX-fed TAC mice relative to sham- orTAC-inoculated TAC mice (FIG. 8C). These included several unidentifiedtaxa from the family S24-7 of the order Bacteroidales, one unassignedtaxon, and four taxa with genus-level identifications, all of which areanaerobic gram-positive bacteria. Of these, the two most significantdifferentially abundant taxa belong to the Bifidobacterium genus, withthe top Bifidobacterium taxon being over 200-fold more abundant in JAXrelative to TAC (p=0.001), and similarly abundant in JAX-fed mice butnot detected at all in TAC-fed TAC mice (p=0.01) (FIG. 3C). Comparisonof relative abundance of all taxa combined belonging to theBifidobacterium genus yielded similar results (FIG. 6A). Given thatinteractions between bifidobacteria and the host immune system have beendescribed previously (Lopez et al. International journal of foodmicrobiology 138, 157-165 (2010).; Ménard et al. Applied andEnvironmental Microbiology 74, 660-666 (2008).; Dong et al. Early humandevelopment 86, 51-58 (2010).; herein incorporated by reference in theirentireties), it was contemplated that members of this genus representone source of the beneficial anti-tumor immune effects observed in JAXmice.

At the sequence level, Bifidobacterium operational taxonomic unit OTU681370 showed the largest increase in relative abundance in JAX-fed TACmice and the strongest association with anti-tumor T cell responsesacross all permutations (FIG. 8D). This bacterium was further identifiedas most similar to B. breve, B. longum and B. adolescentis (99%identity). To test whether Bifidobacterium spp. may be sufficient toaugment protective immunity against tumors, a commercially availablecocktail of Bifidobacterium species was obtained, which included B.breve and B. longum and administered this by oral gavage, alone or incombination with αPD-L1, to TAC 7 recipients bearing established tumors.Analysis of fecal bacterial content revealed that the most significantchange in response to Bifidobacterium inoculation occurred in theBifidobacterium genus (p=0.0009, FDR=0.015, non-parametric t-test), witha 120-fold increase in OTU_681370 (FIG. 9A), indicating that thecommercial inoculum contained bacteria that were at least 97% identicalto the taxon identified in JAX and JAX-fed TAC mice. An increase inBifidobacterium could also be detected by quantitative PCR (FIG. 9B).

Bifidobacterium-treated mice displayed significantly improved tumorcontrol in comparison to non-Bifidobacterium treated counterparts (FIG.8E), which was accompanied by robust induction of tumor-specific T cellsin the periphery (FIG. 8F) and increased accumulation ofantigen-specific CD8+ T cells within the tumor (FIG. 8G and FIG. 9C).These effects were durable for several weeks (FIG. 9D-E).

The therapeutic effect of Bifidobacterium feeding was abrogated inCD8-depleted mice (FIG. 10A), suggesting that the mechanism was notdirect but rather through host anti-tumor T cell responses. Heatinactivation of the bacteria prior to oral administration also abrogatedthe therapeutic effect on tumor growth and reduced tumor-specific T cellresponses to baseline (FIG. 10B-D), suggesting that the anti-tumoreffect requires live bacteria. As an alternative strategy, thetherapeutic effect of B. breve and B. longum strains obtained from theATCC was tested, which also showed significantly improved tumor control(FIG. 11A). Administration of Bifidobacterium to TAC mice inoculatedwith B16 parental tumor cells or MB49 bladder cancer cells also resultedin delayed tumor outgrowth (FIGS. 11, B and C respectively). Oraladministration of Lactobacillus murinus to TAC mice, which was not amongthe overrepresented taxa in JAX-fed mice, had no effect on tumor growth(FIG. 11D) or on tumor-specific T cell responses (FIG. 11E), suggestingthat modulation of anti-tumor immunity depends on the specific bacteriaadministered. Collectively, these data point to Bifidobacterium as apositive regulator of anti-tumor immunity in vivo.

Upon inoculation with Bifidobacterium, a small set of species werealtered in parallel with Bifidobacterium (ANOSIM, p=0.003, FIG. 12A),however, they largely did not resemble the changes observed withJAX-feces administration. Although reductions were observed (˜2-10 fold)in members of the order Clostridiales as well as in butyrate-producingspecies upon Bifidobacterium inoculation, which could point to aninhibitory effect on the regulatory T cell compartment, no differencewas observed in the frequency of CD4⁺ Foxp3⁺ T cells in tumors isolatedfrom JAX and TAC mice (FIG. 12B). Thus, it is unlikely thatBifidobacterium is acting primarily through modulation of the abundanceof other bacteria.

It was next assessed whether translocation of Bifidobacterium wasoccurring into the mesenteric lymph nodes, spleen or tumor, however noBifidobacterium was detected in any of the organs isolated fromBifidobacterium-gavaged tumor-bearing mice (FIG. 12C). It was thusconcluded that the observed systemic immunological effects are occurringindependently of bacterial translocation.

To test whether Bifidobacterium spp may be sufficient to augmentprotective immunity against tumors, we administered a combination offour Bifidobacterium species was administered by oral gavage, alone orin combination with αPD-L1, to TAC recipients bearing 7-day establishedtumors. Bifidobacterium-treated mice displayed significantly improvedtumor control in comparison to non-Bifidobacterium treated counterparts(FIG. 3D), which was accompanied by robust induction of tumor-specific Tcells in the periphery (FIG. 3E) and markedly increased accumulation ofantigen-specific CD8⁺ T cells within the tumor (FIG. 3F). Thistherapeutic effect was completely abrogated in CD8-depleted mice (FIG.3G), arguing that the mechanism was not direct but rather through hostanti-tumor T cell responses. Heat inactivation of the bacteria prior tooral administration also abrogated the therapeutic effect on tumorgrowth and reduced tumor-specific T cell responses to baseline (FIG.6B-D), indicating that the anti-tumor effect requires live bacteria.Administration of Bifidobacterium to TAC mice inoculated with B16parental tumor cells or MB49 bladder cancer cells also resulted indelayed tumor outgrowth (FIG. 6E-F). Oral administration ofLactobacillus murinus to TAC mice, which was not among theoverrepresented taxa in JAX or JAX-fed mice, had no effect on tumorgrowth (FIG. 6G) nor on tumor-specific T cell responses (FIG. 6H),indicating that modulation of commensal bacterial communities throughintroduction of new bacteria in itself does not induce immunity totumors, but rather immunity depends on the specific bacteriaadministered. Collectively, data identify Bifidobacterium as a positiveregulator of anti-tumor immunity in vivo.

To interrogate mechanisms underlying the observed differences in T cellresponses between JAX and TAC mice, we transferred CF SE-labeledSIY-specific 2C TCR Tg T cells into tumor-bearing mice and tested theirproliferation and acquisition of IFN-γ production ex vivo (FIG. 7A).CD8⁺ SIY-specific 2C TCR Tg T cells exposed to tumors in JAX miceexhibited similar expansion in the tumor-draining lymph node as comparedto their counterparts in TAC mice (FIG. 7B). However, they producedmarkedly greater IFN-γ in both the tumor draining lymph node and thespleen of JAX tumor-bearing mice (FIGS. 4A and 4B), suggesting thatsignals upstream of T cells in the JAX environment enhanced acquisitionof T cell effector function. These data indicated an improvement inimmune responses upstream of T cells, at the level of host dendriticcells (DCs). Genome-wide transcriptional profiling of earlytumor-infiltrating DCs isolated from JAX, TAC andBifidobacterium-treated TAC mice was employed (FIG. 13A). In total,there were 761 gene transcripts upregulated by ≥1.5-fold in both JAX andBifidobacterium-treated TAC-derived DCs relative to DCs from untreatedTAC mice (FIG. 4C). Pathway analysis identified cytokine-cytokinereceptor interaction, T cell activation, and positive regulation ofmononuclear cell proliferation as significantly enriched pathways amongupregulated genes (FIG. 4C and FIG. 13B). Many of these genes have beenshown to be critical for anti-tumor responses including those involvedin CD8⁺ T cell activation and costimulation (H2-m2(MHC-I), Cd40, Cd70,Icam 1)) (Mackey et al. Journal of immunology (Baltimore, Md.: 1950)161, 2094-2098 (1998).; Scholer et al. Immunity 28, 258-270 (2008).; Baket al. Journal of immunology (Baltimore, Md.: 1950) 189, 1708-1716(2012).; herein incorporated by reference in their entireties), DCmaturation (Relb, Ifngr2) (Pan et al. Immunology letters 94, 141-151(2004).; Pettit et al. Journal of immunology (Baltimore, Md.: 1950) 159,3681-3691 (1997).; herein incorporated by reference in theirentireties), antigen processing and cross presentation (Tapbp, Rab27a,Slc11a1) (Compeer et al. Frontiers in Immunology 3, (2012).; Jancic etal. Nature cell biology 9, 367-378 (2007).; Stober et al. Infection andImmunity 75, 5059-5067 (2007).; herein incorporated by reference intheir entireties), chemokine-mediated recruitment of immune cells to thetumor microenvironment (Cxcl9, Cx3cl1, Cxcr4) (Kabashima et al. TheAmerican Journal of Pathology 171, 1249-1257 (2007).; Nukiwa et al.European journal of immunology 36, 1019-1027 (2006).; Zhang et al. NewEngland Journal of Medicine 348, 203-213 (2003).; herein incorporated byreference in their entireties) and type I interferon signaling (Irf1,Ifnar2, Oas2, Ifi35, Ifitm1) (Fuertes et al. The Journal of experimentalmedicine 208, 2005-2016 (2011).; Woo et al. Immunity 41, 830-842 10.;herein incorporated by reference in their entireties) (FIG. 4D).Expression of these genes was also strongly induced in murine bonemarrow-derived DCs stimulated with Bifidobacterium in vitro. Takentogether, these data indicate that commensal bacteria-derived (e.g.,Bifidobacterium-derived) signals modulate the activation of innateantigen-presenting cells, which in turn support improved activation oftumor antigen-specific CD8⁺ T cells.

To test whether functional differences in DCs isolated from TAC, JAX andBifidobacterium-treated TAC mice could be sufficient to explain thedifferences in T cell priming observed in vivo, DCs were purified fromlymphoid tissues of naïve TAC, JAX, and Bifidobacterium-treated TAC miceand tested their ability to induce CF SE-labeled CD8⁺ STY⁻ specific 2CTCR Tg T cell proliferation and acquisition of IFN-γ production invitro. DCs purified from JAX and Bifidobacterium-treated TAC miceinduced 2C T cell proliferation at lower antigen concentration comparedto DCs purified from naïve TAC mice (FIGS. 14, A and B). Furthermore, atall antigen concentrations, JAX-derived DCs elicited elevated levels ofT cell IFN-γ production (FIG. 4E and FIG. 14A). Similar effects wereobserved upon oral administration of Bifidobacterium to TAC mice priorto DC isolation (FIG. 4E and FIG. 14A). Taken together, these dataindicate that commensal Bifidobacterium-derived signals modulate theactivation of DCs in the steady state, which in turn supports improvedeffector function of tumor-specific CD8⁺ T cells.

Experiments conducted during development of embodiments hereindemonstrate an unexpected role for commensal microflora (e.g.,Bifidobacterium) in enhancing anti-tumor immunity. These data supportthe idea that one source of inter-subject heterogeneity with regard tospontaneous anti-tumor immunity and therapeutic effects of antibodiestargeting the PD-1/PD-L1 axis may be the specific composition of gutmicrobes, which can be manipulated for therapeutic benefit.

REFERENCES

The following references, some of which are cited above, are hereinincorporated by reference in their entireties.

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1. A method of treating cancer in a human subject comprisingco-administering to the subject an immune checkpoint inhibitor whereinthe immune checkpoint inhibitor is an antibody, and a bacterialformulation comprising bacteria of the genus Bifidobacterium, whereinthe bacterial formulation comprises bacteria selected from the groupconsisting of Bifidobacterium animalis and Bifidobacterium breve. 2.(canceled)
 3. The method of claim 1, wherein at least 90% of thebacteria in the bacterial formulation are of the genus Bifidobacterium.4. (canceled)
 5. (canceled)
 6. The method of claim 1, wherein thebacterial formulation is administered by oral administration.
 7. Themethod of claim 1, wherein the bacterial formulation comprises at least5×10⁶ CFU of bacteria of the genus Bifidobacterium. 8.-18. (canceled)19. The method of claim 1, wherein the immune checkpoint inhibitor isadministered by intravenous injection, intramuscular injection,intratumoral injection or subcutaneous injection. 20.-29. (canceled) 30.The method of claim 1, wherein the immune checkpoint inhibitor isselected from the group consisting of pembrolizumab, pidilizumab,AMP-224, AMP-514, TSR-042, RG-7446, BMS-936559, BMS-936558, STI-1110,MK3475, CT-011, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 andSTI-A1010.
 31. The method of claim 1, wherein the immune checkpointinhibitor is an anti-PD-L1 antibody.
 32. The method of claim 31, whereinthe immune checkpoint inhibitor is selected from the group consisting ofBMS-936559, RG-7446, MEDI-4736, MSB-0010718C and STI-A1010.
 33. Themethod of claim 1, wherein the immune checkpoint inhibitor is ananti-PD-1 antibody.
 34. The method of claim 33, wherein the anti-PD-1antibody is selected from the group consisting of BMS-936558,pembrolizumab, pidilizumab, AMP-224, TSR-042, STI-1110 and MPDL3280A.35. The method of claim 1, wherein the immune checkpoint inhibitor is ananti-CTLA-4 antibody.
 36. The method of claim 1, wherein the immunecheckpoint inhibitor is pembrolizumab and the bacterial formulationcomprises B. breve.
 37. The method of claim 1, wherein the immunecheckpoint inhibitor is MSB-0010718C and the bacterial formulationcomprises B. breve.
 38. The method of claim 1, wherein the immunecheckpoint inhibitor is MSB-0010718C and the bacterial formulationcomprises B. bifidum.
 39. The method of claim 1, wherein the cancer is asolid tumor cancer.
 40. The method of claim 39, wherein the solid tumorcancer is a melanoma.
 41. The method of claim 1, wherein the immunecheckpoint inhibitor is an anti-PD-1, anti-PD-L1, or anti-CTLA-4antibody, and a bacterial formulation comprising bacteria of the genusBifidobacterium, wherein at least 90% of the bacteria in the bacterialformulation are of a single species selected from the group consistingof Bifidobacterium animalis, Bifidobacterium bifidum, andBifidobacterium breve, and wherein the treatment increases CD8+tumor-antigen specific T cells within the cancer.